in bacon, sausage, ham and other cured meats are around. 0.03 mg (g food)-' (Knight .... 0.13 M-NaC1. At this salt concentration, the pale brown, hydrogenase- ...
Journal of General Microbiology (1990), 136, 2067-2076.
Printed in Great Britain
2067
Electron paramagnetic resonance spectroscopic investigation of the inhibition of the phosphorwlastic system of Clostridum sporogenes by nitrite MARTIN J. PAWE,’*LEONARD F. J. WOODS,^ PAULGIBBS~ and RICHARD CAMMACK~ Department of Bwmolecular Sciences, King’s College London, Campden Hill Road, Kensington, London W8 7AH, UK 2MicrobiologySection, Leatherhead Food Research Association, Randalls Road, Leatherhead, Surrey KT22 7RY, UK (Received 25 April 1990; accepted 13 June 1990)
The proposal that nitrite exerts its inhibitory effect on anaerobic bacteria by direct interaction with the ironsulphur proteins of the phosphoroclastic system was investigated. The effects of nitrate, nitrite with or without ascorbate, and nitric oxide on the growth of Clostridium sprogenes in liquid cultures at pH 7.4, on the rates of hydrogen production, and on the activities of the enzymes pyruvate-ferredoxin oxidoreductase and hydrogenase, and of ferredoxin were investigated. In agreement with previous studies, nitrate was the least effective inhibitor of cell growth, and nitric oxide the most effective. Nitrite reductase activity was very low in C. sporogenes,indicating that the presence of external reducing agents would be necessary for the reduction of nitrite to nitric oxide. Inhibition by nitrite was enhanced by ascorbate; 0.5 mM-nitritewith 10 mM-ascorbatestopped growth completely. In partially-purified preparations 4.1 mM-NaNO, and equimolar ascorbate caused complete inactivation of hydrogenaseactivity but only partial (up to 78 %) inactivationof pyruvate-ferredoxinoxidoreductase. This agreed with the loss of hydrogen production observed with nitrite in vivo. Inhibition occurred within 5 min, and was irreversible in each case. Electron paramagnetic resonance (EPR) spectroscopy showed that paramagnetic [Fe(NO),(SR),] species were formed during growth in the presence of nitrite, and were associated with cells. However, the intensity of these EPR signals did not correlate with the inhibition of cell growth. The [4Fe-4S] clusters in ferredoxh were shown by EPR spectroscopy to be resistant to treatment with 3.6mM-NaNO, and 3.6nml-ascorbate. It is concludedthat the eKectsof nitrite on pre-formed iron-snlphurproteins are not convincing as a basis for the lethal effects on bacterial cells.
Introduction Nitrate and nitrite are preservatives used widely to inhibit the growth of clostridia in foods such as meat. The bacteriostatic effect of nitrate operates mainly through reduction to nitrite, which is further converted to other inhibitory species. The effects of these compounds are enhanced by reducing agents, such as ascorbate, which are added to meat products as stabilizers. The maximum NaN02 concentrations used in bacon, sausage, ham and other cured meats are around 0.03 mg (g food)-’ (Knight et al., 1987), which corresponds to approximately 0.4 mnmitrite. Inhibition of clostridial growth by nitrate and nitrite has been widely studied over the past 20 years. These studies have involved a wide range of species and growth media ranging from fluid-broth to meats (Roberts & Ingram, Abbreviations: DTT, dithiothreitol; [Fe-%NO], iron-thiol-nitrosyl complex. 0001-6207
0 1990 SGM
1966; Perigo et al., 1967; Perigo & Roberts, 1968; BairdParker & Baillie, 1973; O’Leary & Solberg, 1976; Woods et al., 1981; Woods & Wood, 1982). The bacteriostatic action of NaN02 in these systems is enhanced when it is reduced by ascorbate (Tompkin et al., 1978a, b ; Pierson & Smoot, 1981). Perigo & Roberts (1968) found that when nitrite was heated with reinforced clostridial medium at pH 6-7, the nitrite disappeared and the medium became more inhibitory to clostridial growth. This is referred to as the ‘Perigo effect’, and is due to the heat-dependent formation by nitrite of more powerful inhibitors, termed ‘Perigo-type factors’ (Larkworthy et al., 1977). Various inhibitory species, such as S-nitrosocysteine and Roussin’s black salt, [Fe,S,(NO),] (Asan & Solberg, 1976), have been isolated from meats after nitrite addition. However, these have not been found in sufficient quantities to account for the levels of inhibition observed.
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M . J. Payne and others
Evidence has been presented that nitrite acts upon metalloproteins which are essential for bacterial growth. Tompkin et al. (1978~)noted that excess iron caused a decrease in the effectiveness of nitrite inhibition of cell growth, and suggested that nitric oxide formed complexes with iron-sulphur proteins such as the clostridial ferredoxins, which are essential for electron transport, enzyme activity and energy production. Iron-sulphur complexes are reactive with nitric oxide, forming a range of iron-thiol-nitrosyl ([Fe-S-NO]) complexes (Butler et al., 1988). The phosphoroclastic system contains three iron-sulphur proteins : pyruvate-ferredoxin oxidoreductase, ferredoxin and hydrogenase. The inhibition of this system would be an effective way of inhibiting spore germination and cell growth. Woods et al. (1981) found that when nitrite was added to a suspension of C. sporogenes cells in glucose medium, it resulted in a decrease in intracellular ATP and an accumulation of pyruvate in the medium. Similarly in C. botulinum cells, nitrite caused a decrease in intracellular ATP (Woods & Wood, 1982). It was concluded that the inhibition of the phosphoroclastic system of C. sporogenes by nitrite involved the reduction of nitrite to nitric oxide, and that inhibition was likely to occur by attachment to the non-haem iron centres present in ferredoxin and pyruvate-ferredoxin oxidoreductase. Pyruvate-ferredoxin oxidoreductase activity was inhibited to a greater extent than ferredoxin activity (Woods et al., 1981). The activity of ferredoxin, in an NADH-linked assay, as electron acceptor from pyruvate-ferredoxin oxidoreductase was unaffected by preincubation with 6 mM-sodium nitrite. The inhibitory action of nitrite on the phosphoroclastic system was not as evident in vitro as in vivo (Woods & Wood, 1982). Angermaier & Simon (1983) studied the hydrogenase and ferredoxin-mediated reduction of aliphatic and aromatic nitro-compounds in clostridia, and proposed that nitrite possibly reacted with cysteine residues with the formation of stable S-nitrosothiols, thereby releasing the iron and sulphur from the clusters. However, Reddy et al. (1983) showed that cells of C. botulinum, after treatment with nitrite, yielded EPR signals with g-values of 2.02-2.04. This type of spectrum is characteristic of low-spin nitrosyl-iron complexes of the general formula [Fe(NO)2(SR),] (McDonald et al., 1965), and has been observed in a variety of biological systems, including animal tissues, after treatment with nitrite (Vanin & Varich, 1981). Reddy et al. (1983) proposed that the formation of these complexes from iron-sulphur proteins and nitric oxide was the mechanism of botulinal inhibition by nitrite in foods. Carpenter et al. (1987) incubated C. botulinum cells with 14.5 mM nitrite, and estimated 94% inactivation of ferredoxin (assayed by monitoring the rate of reduction
of cytochrome c) and 70% inhibition of pyruvateferredoxin oxidoreductase. They stated that it was likely that nitrite exerted its antibtulinal effect by the destruction of iron-sulphur enzymes within vegetative cells. In contrast to the earlier conclusions by Woods et al. (1981) they concluded that nitrite preferentially inhibited ferredoxin in vitro, although pyruvateferredoxin oxidoreductase activity was also inhibited. We have investigated the mode of action of nitrite on the iron-sulphur proteins of the phosphoroclastic system of C. sporogenes. This organism is metabolically similar to the proteolytic strains of C. botulinum and C. perfringens, and therefore is useful as a model to investigate the inhibitory effect of nitrite and derivatives on food-spoilage bacteria. The direct effect of nitrite on the phosphoroclastic system in growing cells was studied by monitoring hydrogen production. The iron-sulphur proteins of the phosphoroclastic system, ferredoxin, pyruvate-ferredoxin oxidoreductase and hydrogenase, were studied in growing cells, in soluble cell extracts, and as the partially-purified proteins. They were incubated with nitrate, nitrite or nitric oxide, and changes in enzyme activities and the formation of EPR-detectable nitrosyl complexes were monitored.
Methods Organism and growth Conditions. Actively growing cultures of
C. sporogenes, strain NCIB 10696, were inoculated (10 ml 1-I) into Oxoid nutrient broth no. 2, containing 1% (w/v) glucose and 5 ml I-' of 10% (w/v) sodium thioglycollate, and incubated at 37 "C;Cell growth was monitored as optical density at 550 nm. Cells were harvested by centrifugation after incubation for a further 20 h.
Inhibitors. Nitrite was autoclaved in 10 1 of standard Oxoid nutrient broth no. 2, prior to the addition of glucose and thioglycollate. The nutrient medium was then inoculated with 10ml actively growing clostridia 1-'. Cells were incubated at 37 "C for 24 h before harvesting. Nitric oxide was added to cultures as a saturated solution. Water was thoroughly saturated with oxygen-free argon before bubbling with nitric oxide on ice under anaerobic conditions, to avoid oxidation of NO and consequent acidification, Purijication of proteins Soluble cell extracts. Extracts were prepared in an anaerobic glovebox (Miller Howe Ltd) in an atmosphere of nitrogen (
