ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 347, No. 2, November 15, pp. 271–274, 1997 Article No. BB970343 Superoxide Imposes Leakage of Sulfite from Escherichia coli Ludmil Benov and Irwin Fridovich 1 Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 Received July 1, 1997, and in revised form August 8, 1997 on the pathway from sulfate. This intermediate accu- Escherichia coli, which lacks the cytosolic superox- mulated in the medium until it reached a concentration ide dismutases, exhibits several nutritional auxotro- sufficient to support growth. The need for cysteine and phies when growing aerobically. The cysteine/methio- methionine was thus a bradytrophy rather than an ab- nine requirement, which is one of these, was previously solute auxotrophy. The intermediate, whose leakage shown to be due to leakage from the cells, and accumu- accounted for that bradytrophy, appeared to be sulfite lation in the medium, of a metabolic intermediate on on the basis of a bioassay which utilized mutants with the biosynthetic route to these amino acids. The paren- specific defects in the cysteine operon. Furthermore, tal strain does not significantly accumulate this com- addition of sulfite to the medium did facilitate the aero- pound. It is now shown that treatment with alkaline bic growth of the sodA sodB strain. However, attempts cyanide releases sulfite from this compound, a prop- to chemically demonstrate sulfite in the conditioned erty shared by a-hydroxy sulfonic acids (carbonyl – bi- medium failed, as did attempts to recover sulfite added sulfite adducts). Since E. coli accumulates carbonyl to that medium (8). compounds in the growth medium, it appears likely We now report that treatment with alkaline cyanide that the sulfitogenic compounds accumulated by the (9) allows the recovery of sulfite from the conditioned sodA sodB strain are a-hydroxy sulfonic acids.  1997 medium. It is further shown that carbonyl compounds Academic Press prevent the recovery of sulfite in the absence of the CN 0 Key Words: cysteine, auxotrophy of; sulfite, leakage treatment and that carbonyl compounds accumulate in of; superoxide, effect of; carbonyls, leakage of; cryptic the conditioned medium. These and related results are sulfite. reported below. MATERIALS AND METHODS Escherichia coli, with insertional defects in the genes Bound sulfite was liberated by treating 1.0-ml samples with 0.25 coding both for the inducible MnSOD (sodA) 2 and the ml of 2.5% NaCN containing 50 mM EDTA and, after adjusting the constitutive FeSOD (sodB), exhibit a number of dioxy- pH to between 10 and 11 with 0.1 ml 1.0 M NaOH, heating to 50°C for 30 min in covered tubes (9). Sulfite released in this procedure gen-dependent phenotypic deficits. These include auxo- was assayed by adding 4.0 ml of fuchsin reagent (10) at room temper- trophies for certain classes of amino acids (1), enhanced ature, clarifying by filtration through 0.22 mM filters, and reading mutagenesis (2), and stationary phase death (3). The at 575 nm. The time elapsed between adding the fuchsin reagent requirement for branched chain amino acids has been and measuring A 575 nm was 5 min. Calibration with freshly prepared explained on the basis of a [4Fe – 4S]-containing dehy- Na 2 SO 3 was performed each day. Carbonyl compounds were assayed by treating 1.0-ml samples with 1.5 ml of 0.1% 2,4-dinitrophenyl- dratase on the biosynthetic pathway to these amino hydrazine in 2.0 N HCl, incubating for 5 min at 25°C, and then acids. This enzyme, the dihydroxy acid dehydratase, is adding 2.5 ml of 2.5 N NaOH with vigorous agitation. Six minutes rapidly inactivated by O 0 2 (4–7). after alkalinization samples were clarified by centrifugation and The sodA sodB strain also requires sulfur-containing A 540 nm was measured against a blank (11). amino acids. This was shown to be due to a dioxygen- Bacteria. The strains of E. coli used were JI132 which bears in- dependent leakage from the cells of some intermediate sertional defects in sodA and sodB, and AB1157, which is the paren- tal strain (12). Cultures were grown overnight at 37°C in aerated LB medium, which contained (per L) 10 g Bacto-tryptone, 5 g yeast 1 To whom correspondence should be addressed. Fax: (919) 684- extract, and 10 g NaCl, with pH adjusted to 7.0 with K 2 HPO 4 . The overnight cultures were harvested by centrifugation and the cells 8885. 2 Abbreviations used: JI132, sodA sodB E. coli; AB1157, the paren- were thrice washed with M9 salts (13). They were then suspended in 200 vol of M9 salts supplemented with 100 mg/L of each of the tal strain of E. coli; SOD, superoxide dismutase. 271 0003-9861/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved. AID ABB 0343 / 6b43$$$$81 10-15-97 22:50:13 arcal