Characterization of anaerobic sulfite reduction by Salmonella typhimurium and purification of the anaerobically induced sulfite reductase

Abstract

Mutants of Salmonella typhimurium that lack the biosynthetic sulfite reductase (cysI and cysJ mutants) retain the ability to reduce sulfite for growth under anaerobic conditions (E. L. Barrett and G. W. Chang, J. Gen. Microbiol., 115:513-516, 1979). Here we report studies of sulfite reduction by a cysI mutant of S. typhimurium and purification of the associated anaerobic sulfite reductase. Sulfite reduction for anaerobic growth did not require a reducing atmosphere but was prevented by an argon atmosphere contaminated with air (<0.33%). It was also prevented by the presence of 0.1 mM nitrate, which argues against a strictly biosynthetic role for anaerobic sulfite reduction. Anaerobic growth in liquid minimal medium, but not on agar, was found to require additions of trace amounts (lo-7 M) of cysteine. Spontaneous mutants that grew under the argon contaminated with air also lost the requirement for 10-7 M cysteine for anaerobic growth in liquid. A role for sulfite reduction in anaerobic energy generation was contraindicated by the findings that sulfite reduction did not improve cell yields, and anaerobic sulfite reductase activity was greatest during the stationary phase of growth. Sulfite reductase was purified from the cytoplasmic fraction of the anaerobically grown cysI mutant and was purified 190-fold. The most effective donor in crude extracts was NADH. NADPH and methyl viologen were, respectively, 40 and 30% as effective as NADH. Oxygen reversibly inhibited the enzyme. Two highmolecular-weight proteins separated by gel filtration (Mr 360,000 and 490,000, respectively) were required for maximal activity with NADH. Indirect evidence, including in vitro complementation experiments with a cysG mutant extract, suggested that the 3609000-Mr component contains siroheme and is the terminal reductase.This component was further purified to near homogeneity and was found to consist of a single subunit of molecular weight 67,500. The anaerobic sulfite reductase showed some resemblance to the biosynthetic sulfite reductase, but apparently it has a unique, as yet unidentified function.Although much is known about sulfite reduction by the sulfate-reducing bacteria (1, 23, 28), very little is known about the anaerobic production of H2S from sulfite by members of the family Enterobacteriaceae. The ability to perform this reaction is routinely used in diagnostic laboratories to differentiate Salmonella species (H2S positive) from Escherichia coli (H2S negative). We have hypothesized that the enzymes involved may explain the observation that cysI and cysJ (biosynthetic sulfite reductase) mutants of S. typhimurium behave as prototrophs under anaerobic conditions (2), whereas E. coli cysI and cysJ mutants are auxotrophs under both aerobic and anaerobic conditions (E. L. Barrett, unpublished findings). Like E. coli, S. typhimurium mutants defective in cysG, which is required for synthesis of the siroheme prosthetic group (25), do not produce H2S from sulfite and do not behave as prototrophs under anaerobic growth conditions. The...

Sequence analysis and expression of the Salmonella typhimurium asr operon encoding production of hydrogen sulfide from sulfite

“…Firstly, it produces significant quantities of free hydrogen sulfide from sulfite, a property which, among the Enterobacteriaceae, is unique to the genera Salmonella and Edwardsiella (2,41). Our studies of H2S production from sulfite by S. typhimurium have shown that it is strictly anaerobic (21), genetically distinct from its biosynthetic analog (21,24), linked to NADH rather than NADPH oxidation (21), and regulated by available electron acceptors rather than by cysteine (6,21,24). Thus, it is essentially a dissimilatory process.…”

Identification and cloning of genes involved in anaerobic sulfite reduction by Salmonella typhimurium

“…Salmonella species, but not E. coli, are known to reduce sulfite to hydrogen sulfide (5,19). This reduction appears to be dissimilatory rather than assimilatory, because it is negatively regulated by the availability of the electron acceptors oxygen and nitrate rather than by the availability of cysteine (5,11). We have hypothesized that the dissimilatory sulfite reductase activity may be the explanation for the cysI and cysJ mutant anaerobic prototrophy.…”

“…They were purified on green indicator plates (14). The 16,000 x g for 20 min, and the clear supernatant was used immediately for the enzyme assay as previously described (11). Protein was determined by the Bradford method (3).…”

Mutations that affect the regulation of phs in Salmonella typhimurium

“…probably in or near the phs promoter operator region; and (2) that the gene in which the original insertion was isolated is not autoregulated. Since the phs locus appears to play a part in the regulation of thiosulphate reductase, we assayed thiosulphate reductase activity, using a previously described method (Hallenbeck et al, 1989), and again found no difference between the diploid strains described above and the phs-haploid strain EB222 (results not shown).…”

“…However, unlike E. coli, under anaerobic conditions S. typhimurium produces H2S from either thiosulphate [via a thiosulphate reductase (Barrett & Clark, 1987;Clark & Barrett, 1987a)l or from sulphite [via an anaerobically induced sulphite reductase (Hallenbeck et al, 1989)l. Previous studies have defined a genetic locus, phs, that appears to be essential for anaerobic thiosulphate reduction in vivo and for in vitro thiosulphate reductase activity (Clark & Barrett, 1987a, b;Voll et al, 1974Voll et al, , 1979.…”