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Iron metabolism in bifidobacteria
Int. Dairy Journal 6 (1996) 905-919 Copyright PII:
SO958-6946(96)00003-9
0
1996 Elsevier Science Limited
Printed in Ireland. All rights reserved 095%6946/96/$15.00 + 0.00
ELSEVIER
Review Article Iron Metabolism in Bifidobacteria
Anatoly Bezkorovainy, * Eva Kot, Robin Miller-Catchpole, George Haloftis & Sergey Furmanov Department
of Biochemistry, (Received
Rush Medical
15 September
College,
1995; accepted
Chicago,
IL 60612-3833,
23 December
USA
1995)
ABSTRACT Btjidobacteria are Gram-positive, anaerobic microaerophilic rods that are capable of internalizing ferrous iron at pH 5.0 and 6.5 when assayed in a post-logarithmic growth phase. Dependent upon iron concentration, iron uptake is most efficient in cells grown in a metal-depleted medium. There are two iron-uptake systems: one operating at low outside iron concentrations (1 to 20 PM),' and one operating at higher concentrations (up to 400 PM). Iron is located largely on the surface and in the cell-solublefraction at low iron concentrations. At high levels, iron is associated mostly with the insoluble (particulate) fraction. While the soluble iron is in the ferrous state, most of the insoluble iron is Fe(OH)s. It has been proposed that bijidobacteria contain an intracellular ferroxidase that oxidizes internalized Fe’+ to Fe(III) using oxygen as an electron acceptor. In B. thermophilum, the K, of this ferroxidase is 518 pM. Iron uptake by btjidobacteria depends on sugar metabolism, though minor amounts of iron are taken up in the absence of a carbon source. Iron uptake does not involve any siderophores or other carriers. The iron is apparently transported by a divalent metal permease, which requires a functioning ATPase and a proton gradient. There are many similarities between the mode of iron transport in btjidobacteria and other Gram-positive bacteria such as S. mutans and L. acidophilus, and between iron transport in btjidobacteria andmanganese transport in L. plantarum. Future work on metal transport in btjidobacteria is likely tofocus on the identification and isolation of proteins involved in iron transport and oxidation. Clinical implications of the iron uptake phenomenon in btjidobacteria are also promising areas of ,future research. Copyright 0 1996 Elsevier Science Limited *Author
to whom correspondence
should be addressed 905
906
A. Bezkorovainy et al.
INTRODUCTION Species of the genus Bifidobacterium are normally encountered in most if not all animal gastrointestinal tracts. These organisms are beneficial to humans, especially in newborns, where they maintain colonic pH at about 5 and prevent the growth of pathogens (Bullen et al., 1976). The discovery of new bifidobacterial species is an ongoing process. As of 1992, some 28 species have been identilied (Biavati et al., 1992). Though such species differ to some extent from each other morphologically and physiologically, all are Gram-positive rods with occasional bifid forms, catalase-negative, non-sporeforming, and fermenting glucose to acetate and lactate via a unique ‘bitidus’ pathway. An enzyme of this pathway, phosphofructoketolase, is used routinely to distinguish bitidobacteria from other microorganisms (Scardovi, 1986). Numerous health benefits have been ascribed to the consumption of bilidobacteria; these include alleviation of gastrointestinal infections (especially in young children), constipation and diarrhea, antitumor action, lowering of serum cholesterol and improving the immune response (Kurmann & Rasic, 1991; Alm, 1991). There has been considerable interest in determining whether or not such health claims can be rigorously confirmed (Sanders, 1993). The means for administering bifidobacteria to human beings for therapeutic or prophilactic purposes have been various dairy products, especially fermented milk and yogurt (Robinson, 1991; Hughes & Hoover, 1991; Ishibashi & Shimamura, 1993). In fact, many yogurt brands in Europe, the US and especially Japan are manufactured using bifidobacteria as one of the starter culture types. There is interest in determining whether or not bifidobacteria can significantly improve the tolerance of milk by lactose-intolerant individuals (e.g. Passerat & Desmaison, 1995), and there is concern about the relatively short periods of time that bifidobacteria can survive upon storage in various types of milk (Lee & Wong, 1993; Hughes & Hoover, 1995). Metabolism, growth characteristics and the physiological peculiarities of various bitidobacterial strains are thus intimately associated with manufacture, marketing and utilization of dairy products (Hughes & Hoover, 1991). Many beneficial effects of bifidobacteria and lactic acid bacteria in humans have been attributed to their production of organic acids (Bullen et al., 1976); however, such effects could also be the result of an efficient sequestration of iron by these bacteria, thus making it unavailable to pathogens (Bezkorovainy & Solberg, 1989). This phenomenon has often been referred to as nutritional immunity, and has usually been associated with the iron-binding properties of transferrin and lactoferrin (Weinberg, 1984). Our laboratory has been concerned with iron metabolism in bifidobacteria for a number of years. The species studied have been BiJidobacterium biJidum var. pennsyfvanicus (ATCC 11863), Bzjidobacterium breve (ATCC 15700), and Bifidobacterium thermophilum (ATCC 25866). The first two are of human origin, whereas the latter was initially isolated from bovine rumen. In our studies, we have been concerned primarily with the metabolism of ferrous iron, the form that would be available to these bacteria in their normally anaerobic habitats (Kammler et al., 1993). Very little is known about iron transport in Gram-positive prokaryotic organisms in general. Thus, review articles by Crichton & Charloteaux-Wauters (1987)
Iron metabolism in bifidobacteria
901
and by Briat (1992) make no mention of iron transport in these organisms, and that by Guerinot (1994) does so only in passing. On the other hand, iron transport in Gram-negative bacteria has been studied and documented very thoroughly and has been the subject of many reviews (e.g. Neilands, 1981; Crosa, 1989). To our knowledge, therefore, bifidobacteria appear to be the only Grampositive genus, where iron transport has been under investigation in some detail, and this has taken place in our laboratory. This review is being offered firstly as a means of calling the attention of the scientific community to this relatively uncharted area or research, and secondly, to summarize the work done in our laboratory in the past 12 years. Our objective is to present evidence that iron is a bilidobacterial growth factor, to argue in support of a ferrous iron transport hypothesis in bifidobacteria, to offer a general mechanism of such transport, and to identify the nature of accumulated iron. Comparisons between iron transport in bifidobacteria and iron and other metal transport in other Gram-positive organisms are also presented.
IRON AS AN ESSENTIAL
NUTRIENT
IN BIFIDOBACTERIA
Like most organisms, bifidobacteria require iron for growth. This was demonstrated by growing B. bifidum in the so-called modified Norris medium (Poupard et al., 1973) in the presence of various iron chelators such as EDTA and bipyridyl (predominantly ferrous iron chelators) and EDHA, 2,3_dihydroxybenzoate, desferrioxamine, 8-hydroxyquinoline and nitrilotriacetate (predominantly ferric iron chelators). The ferrous iron chelators are better growth inhibitors than the ferric ones, and such inhibition can be reversed by the addition of Fe*+ and to a lesser extent by ferric citrate (Bezkorovainy & Topouzian, 1983). Growth of bifidobacteria can be also inhibited by various divalent transition metals and this too can be relieved by the addition of Fe2+ (Topouzian et al., 1984). The mechanism for this effect is most likely based on a direct or indirect competition for a divalent metal transporter (permease) between such metals and iron. The (non)requirement of iron for the growth of lactic acid bacteria, another group of Gram-positive organisms, is more obscure, since at least one of them, Lactobacillus plantarum 14917, does not require iron for growth, but instead requires manganese (Archibald, 1983).
FERROUS
IRON UPTAKE BY BIFIDOBACTERIA
The typical iron uptake experiment involves the incubation of post-logarithmic cell suspensions (A6i0 = 1.2) with 1 to 400pM 59Fe2+ in the presence of ascorbate (as an antioxidant; Fe2+: ascorbate = 5:3), or tagged ferric citrate at pH 5.0 or 6.5 using either 0.1 M acetate buffer (pH 5.0) or 3,3_dimethylglutarate buffer (pH 5.0 and pH 6.5) in air or at low pOZ. In addition, the uptake medium, termed modified Hanks solution (mHanks; Bezkorovainy et al., 1986a), contains 0.4 g KCl, 8 g NaCl, 0.14 g CaC12 and 2 g lactose or glucose per liter. Incubations are carried out at 37°C or in an ice-water bath at t+l”C with shaking. At various times (up to 120 min), five-ml samples are withdrawn, cooled on ice and centrifuged at 4°C. The cells are then washed with ice-cold 0.1 M acetate buffer at pH
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A. Bezkorovainy
et al.
5.0 and counted in a gamma-counter to determine the amount of iron associated with them. The quantity of cells counted represents 2.75 mg of dry weight. Lactic acid production is measured throughout the incubation (uptake) process to assess cell viability. The extent of ferrous iron accumulation by bitidobacteria depends upon their nutritional history. When grown on the regular TPY medium (Scardovi, 1986), little if any ferrous iron is accumulated. However, when exogenous metal salts are omitted, the cells take up ferrous iron readily (Fig. 1) (Kot & Bezkorovainy, 1991). A metal-poor medium, termed modified TPY (mTPY), was thus routinely used to grow bitidobacteria for iron uptake studies. A series of such experiments is illustrated in Fig. 2. It shows that lactic acid is produced as iron is accumulated, that the extent of iron accumulation depends on its concentration in the medium, and that at tM”C, there is little if any iron accumulation. Figure 3 illustrates that a carbon source is important for iron uptake, though some uptake occurs in the absence of a sugar (Bezkorovainy et al., 1986a, b; Bezkorovainy & Solberg, 1989). When cells are heated at 80°C for 15 min, the ability to accumulate iron is lost (Bezkorovainy et al., 1986a). There are three reasons why ferrous iron may become associated with bacterial cells in the above experiments: it can become oxidized by air and precipitate as Fe(OH)s, the presence of the antioxidant notwithstanding; it could become
180 160 140 120 100 80 60 40 20
100
200
300
400
Felin medun (pbll
Fig. 1. Iron accumulation by Bifidobacterium thermophilum as a function of ferrous iron concentration in the mHanks medium and its nutritional history. All incubations were carried out at 37°C for 60 min. Curve A refers to cells grown in the regular TPY medium (Scardovi, 1986); curve B refers to cells grown at [Fe’+]= 1OpM (in modified TPY medium); and curve C refers to cells grown at [Fe”] =5.5 pM (chelated modified TPY medium) (from Kot & Bezkorovainy, 1991).
Iron metabolism in bifidobacteria r
10
20
40
60
60
100
20
120
40
60
60
100
120
Time (min.)
lime (min.)
Fig. 2. Ferrous iron uptake by Bfidobacterium breve. Solid lines represent iron uptake, broken lines, lactate production. Crosses indicate &4”C, whereas circles indicate 37°C. Frame A indicates experiments with 65.1 pM Fe2+, B indicates experiments with 11.4 pM Fe’+. All experiments were performed at pH 5.0 (Bezkorovainy & Solberg, 1989).
bound to the cell surface; or it could become internalized by the cell. Air oxidation of Fe2+ can be eliminated as a possibility by performing the uptake studies at OG4”C and/or with heated cells. If some of the iron is precipitated as Fe(OH)3, it should co-centrifuge with the heated cells or with the cells used at cold
temperatures and one could then detect substantial amounts of iron associated with the cells. This does not occur. Moreover, when ferrous iron is incubated under the above conditions in the absence of bacteria and the amount of ferrous iron remaining is measured by the ferrozine reaction as a function of time, there is little if any loss of ferrous iron at both pH 5.0 and 6.5 (Bezkorovainy et al., 1988). Bifidobacteria do not produce hydrogen peroxide (Kot et al., 1995a), and lactate
u -360 -200
0" a -5 %g Et
-100
2 : J
30
60
120
Time (min.)
by B$dobacterium breve. Solid lines represent iron uptake, broken lines, lactate production. -x-x- are controls; ~ o-o are lactose-deprived cells; -o-e-- are lactose-deprived cells to which lactose was added (2 mg mL-‘) after 30 min of incubation with iron. Ferrous iron concentration was 129 pM in all experiments (from Bezkorovainy & Solberg, 1989).
Fig. 3. Effect of lactose on iron uptake and lactate production
910
A. Bezkorovainy et al.
produced by the organism, while promoting ferrous iron oxidation to some extent, sequesters the ferric iron produced and makes it unavailable for binding to the bacteria (Kot et al., 1995b). The next issue is whether or not surface binding of ferrous iron could account for iron accumulation by the cells. This can be explored by washing the iron-loaded cells with ice-cold 2 mM FeS04 and/or 2-4 mM EDTA in 0.1 M acetate buffer at pH 5.0. At this low temperature, the cellular membrane should be rigid and impermeable to any internalized iron while surface-bound iron should be washed off. Approximately 10% of the accumulated iron is normally lost by such washes (Bezkorovainy & Solberg, 1989), indicating that surface binding of ferrous iron occurs only to a minor extent. Having thus eliminated non-specific precipitation of Fe(OH)3 and surface binding of ferrous iron as major causes of ferrous iron accumulation by bifidobacteria, it becomes clear that under the conditions used in our laboratory most of the iron taken up is internalized by the cells. Further evidence of iron internalization by bitidobacteria is provided by the behavior of their protoplasts and by the effect of calimycin on the process of iron uptake. When protoplasts are prepared from iron-preloaded B. thermophilum cells, only minor losses of iron are observed, indicating that the cell wall does not serve as a major binder of iron. The protoplasts themselves cannot internalize iron, but do, instead, bind large quantities of iron on their surface. This conclusion is reached because iron accumulation by protoplasts is almost instantaneous, most if not all of the iron acquired can be eluted by 2 mM FeS04, and iron accumulation is equal at O-4” and 37°C (Kot et al., 1993a). Calimycin (A 23187) is a divalent metal ionophore, which works well with ferrous iron (Baker et al., 1984; Egyed & Saltman, 1984). When used with bifidobacteria in iron uptake experiments at concentrations of l-2 pM, iron accumulation is completely inhibited (Topouzian & Bezkorovainy, 1986; Bezkorovainy & Solberg, 1989). Such small ionophore concentrations could not bind and therefore inhibit iron uptake when the latter concentrations are 50-200 pM. Its action must therefore involve the short-circuiting of iron gradients that are formed between the cell interior and its environment. Ferrous iron accumulation by bifidobacteria takes place over a rather wide range of its concentrations (l-400 ,uM). At least two iron uptake systems are present in B. breve: the high affinity system, which operates at low iron concentrations (l-20 PM); and a low affinity system that operates at high iron concentrations (5&400 PM). The Km’s of the two systems are 35 and 86 pM, respectively (Bezkorovainy 8z Solberg, 1989). The uptake of ferric iron by bifidobacteria is very low. When ferric iron is presented to the cells as the citrate complex, little if any is accumulated. When ascorbate, a reducing agent, is added, iron accumulation resumes (Bezkorovainy, 1984). The spent bacterial growth medium, as well as the insoluble bacterial fraction obtained following sonication, are able to reduce ferric to ferrous iron using NADH as a reducing agent (Bezkorovainy et al., 1986b). This indicates the presence of a ferrireductase on the cell surface, whose function may be to bolster iron uptake when ferric but not ferrous iron is present in the growth medium. A similar situation is observed in Streptococcus mutuns (Evans et al., 1986).
911
Iron metabolism in bifidobacteria TABLE 1
Effects of Various Metal Ions and Metabolic Inhibitors on Ferrous Iron Uptake by Bilidobacteria” Group
Ion or compound
Effect
Metals
Zn2 ’ co2 + Mg2’ Ca2 ’
Inhibits Inhibits Inhibits No effect
Chelators
Bipyridyl Citrate Desferrioxamine EDHA EDTA Nitrilotriacetate
Inhibits Inhibits Inhibits Inhibits Inhibits Inhibits
lonophores
Calimycin (A23 187) CCCP Nigerisin Valinomycin Valinomycin + nigerisin
Inhibits Inhibits Stimulates Stimulates Inhibit
ATPase inhibitors
DCCD Vanadate
Inhibits Inhibits
1987); “Summarized from Bezkorovainy et a/. (1986b, Bezkorovainy & Solberg (1989); Topouzian & Bezkorovainy (1986).
THE MECHANISM
OF FERROUS IRON UPTAKE BY BIFIDOBACTERIA
The mechanism of ferrous iron uptake by bifidobacteria was investigated by using various types of metabolic inhibitors: divalent cations; metal chelators; iono hores; and enzyme/transport inhibitors (Table 1). Only one metal ion, P inhibits ferrous iron uptake competitively. The mode of action of the co* other; cannot be easily classified into any categories (Bezkorovainy et al., of 200 pM and 1987). Of special interest is Mg2+, which at concentrations higher is able to completely inhibit iron uptake by B. thermophilum. When the Mg*’ is removed from the assay medium, iron uptake resumes. Mg*’ has no effect on lactate production. Ca* + , on the other hand, has a slight stimulatory effect on iron uptake (Kot & Bezkorovainy, 1993a). It may be proposed that ferrous iron and certain other divalent cations share common permeases (transporters) on bifidobacterial plasma membranes. A number of ionophores have either a positive or a negative effect on iron uptake by bifidobacteria. The proton ionophore, CCCP, inhibits iron uptake, while valinomycin and nigericin, when used individually, stimulate iron uptake. When combined, on the other hand, valinomycin and nigericin have an inhibitory effect. ATPase inhibitors such as DCCD and vanadate have inhibitory effects
912
A. Bezkorovainy et al.
(Table 1). Since the transport of nutrients into bacterial cells depends on pH (proton) gradients and on membrane potentials (e.g. Futai & Tsuchiya, 1987), and since both can-be produced by the action of ATPases, we conclude that both the proton gradient and membrane potential are important in forcing the iron into the bifidobacterial cell interior (Bezkorovainy et al., 1987). Others, on the basis of the CCCP and DCCD data, had concluded that the proton gradient was responsible for the uptake of manganese by L. plantarum (Archibald, 1986). In the absence of glucose, when ATPase activity is minimal and protons are not being extruded from the cell interior, ferrous iron uptake is still taking place, though at drastically lowered levels. It is likely that the membrane potential is the driving force under such circumstances (Kot et al., 1993b). Membrane potential is also the driving force for siderophore-bound iron uptake in certain fungi (Huschka et al., 1983). Metal chelators are excellent iron uptake inhibitors rather than iron carriers for bifidobacteria. Such chelators can often act as iron suppliers in other bacteria, e.g. citrate for manganese (Archibald, 1984) and EDHA for iron in some Grampositive and negative organisms (Salamah, 1992). In the case of EDHA action on Gram-negative organisms, one can, in fact, postulate that siderophores are the actual iron carriers and able to retrieve iron from EDHA. The hypothesis that no classical siderophores are involved in iron transport into bifidobacteria is thus strengthened by the observed action of chelators: since siderophores have extremely high association constants for iron (Neilands, 1981) one could not expect relatively mild chelators such as citrate, bipyridyl or nitrilotriacetate to successfully compete with the carrier siderophores for iron and inhibit their action. On the other hand, these chelators are apparently able to withhold iron from the iron permease (Topouzian & Bezkorovainy, 1986). In summary, iron uptake by bitidobacteria takes place via an electrogenic pump mechanism using a permease that may be common to several divalent cations. No siderophore or other carrier is apparently involved.
’ THE NATURE
OF ACCUMULATED
IRON
Iron associated with bifidobacteria following a typical ferrous iron uptake experiment can be divided into three pools: surface-bound iron, which accounts for about 10% of total iron accumulated (see above); soluble iron in the cell cytosol; and iron associated with the insoluble cell fraction, such as the cytosol side of the plasma membrane or another intracellular structure. Intracellular distribution of iron, i.e. its partition between the soluble and insoluble pools, can be accomplished by disrupting the FeSOd-washed cells by sonication or the French pressure cell, centrifugation, and determining iron in the soluble and insoluble fractions. Further, the insoluble fraction can also be washed with 2 mM FeS04 to sub-partition that pool into elutable ferrous iron and non-elutable iron. In B. thermophi(um, the amount of iron found in the insoluble fraction increases as total cellular iron increases; at the same time, iron that is elutable by FeS04 decreases. At low cellular iron levels, most of the internalized iron can be mobilized from the cells by suspending them in iron-free buffers. As cellular iron increases, percent of mobilizable iron decreases (Bezkorovainy et al., 1988; Kot & Bezkorovainy, 1993b).
913
Iron metabolism in bifidobacteria
The question now arises as to the chemical nature of the iron pools described above. Iron in the soluble cell fraction is in the ferrous state: it is easily chelated by ion exchangers and gives a typical color with ferrozine (Kot et al., 1994). Iron bound to the particulate fraction and elutable with 2 IIIM FeS04 is most likely ferrous iron bound to membrane fragments on their cytosol side. The non-elutable particulate fraction-bound iron is most likely in the ferric form. It can be solubilized with a sorbitol-mannitol solution and such extracts absorb at 650 nm, which is typical of ferric iron complexes with polyalcohols (Schneider et al., 1982). The intracellular deposition of ferric iron following ferrous iron uptake can be further investigated by performing iron uptake experiments at low p02 (53 mm Hg). Typical results of such experiments, shown in Table 2, indicate that iron uptake by cells is higher in air than at low p02 and that the distribution of iron between the soluble and insoluble pools of the cells favors the insoluble fraction in air. Glucose amplifies this effect (Kot & Bezkorovainy, 1993b; Kot et al., 1994). The apparent intracellular oxidation of ferrous iron to the ferric form resulting in the deposition of the insoluble Fe(OH)s and its polymers in the cell can be traced to the particulate fraction, which is able to insolubilize some 50% more iron in air than at low pOz (Kot et al., 1994). It is proposed that the particulate fraction of bifidobacteria contains a ferroxidase, for which a &, of 5 18 f 130 pM with respect to Fe*+ can be calculated. This is confirmed in part when very low amounts of ferrous iron (l-l 1 PM) are used in iron uptake experiments, which are well below the Km: the low ~02 atmosphere has no effect, the ratio of insoluble to soluble cellular iron remaining the same at about 0.25 (Kot et al., 1994). We do not believe that the putative intracellular ferroxidase bears any relationship to the ferrireductase described above. The latter appears to be a dehydrogenase using NADH as a substrate, while the ferroxidase is a true oxidase using oxygen as a substrate. The ferrireductase appears to be located on the cell TABLE 2
Uptake of Iron by LMidobacteria at Low ~02 and in Air, and the Effects of Glucose. Iron Concentration
was 192 f 11.2 pM, and Incubations pH 5.0”
Microorganism
Atmosphere
B. thermophilum
Bhreve
were Carried Out at 37°C for 60 min at
Glucose (mM)
Fe2 + uptake (nmol/pellet)b
Distribution ratio’
low poz low po* air air
0.0 11.1 0.0 11.1
53.1 166 64.7 221
0.337 0.404 0.561 1.55
low po* low poz air air
0.0 11.1 0.0 11.1
41.4 135 120 312
0.268 0.243 0.844 1.67
“Adapted from Kot et al. (1994). ‘A pellet corresponds to 2.75 mg dry weight. ‘Iron in the particulate-(insoluble) fraction to that in the soluble cell fraction.
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A. Bezkorovainyet al.
surface and its mission is to generate Fe’+ from Fe(II1) for internalization purposes. The ferroxidase is intracellular and its function is apparently to remove potentially toxic quantities of Fe2+ from the cell interior. Having established that iron is internalized by bifidobacteria via an electrogenic pump mechanism and having defined the bacterial iron pools, one may now attempt to estimate the level of iron concentration by the cells with respect to their environments. Using data from Table 1 of Kot et al. (1994) we find, after correcting for surface binding of iron, that in the presence of glucose, B. breve concentrate iron some 106-fold in the presence of air and 70-fold at low p02. In the absence of glucose, the respective figures are 6%fold and 20-fold. These calculations are based on the determination that bifidobacteria have a cellular volume of 3.25 mL g-’ dry weight (Veerkamp, 1977). This concentration effect is brought about firstly by an active pumping mechanism and secondly by the fact that the internalized ferrous iron becomes oxidized to the insoluble ferric form, Proposed iron movements in the bifidobacterial cell are illustrated in Fig. 4. Note that two forms of iron are associated with the inner side of the membrane: ferrous and Fe(OH)s. It is assumed that the nutrient sugars enter the cells in symport with protons. It is also assumed that ferric iron does not enter the cell directly, but is, instead, reduced to the ferrous form first.
METAL UPTAKE BY VARIOUS GRAM-POSITIVE
BACTERIA
There is a dearth of information regarding iron and other metal transport in Gram-positive bacteria. Nevertheless, some organisms have been investigated to a sufficient extent to provide a point of reference allowing comparison with bilidobacteria. Of special interest is the metal transport in Streptococcus mutans and certain lactic acid bacteria. S. mutans has been implicated in the causation of dental decay. This organism
Glucose, lactose Glucose/Lactose
Cell membrane
+ AcetIc 8 lactic acids
Fig. 4. Proposed scheme for iron movements in a typical bifidobacterial
cell.
Iron metabolism in bifidobacteria
915
takes up iron presented in the ferric form only after reduction to the ferrous form by a membrane-bound reductase. Ferrous iron is taken up much more readily than the ferric form. As is the case with bitidobacteria, iron chelators are able to prevent the internalization of iron. Further details of iron transport in this organism have not been investigated (Evans et al., 1986). Iron metabolism in various lactic acid bacteria is more complicated than it is in bilidobacteria because many such organisms produce hydrogen peroxide in the presence of oxygen (Condon, 1987). Hydrogen peroxide rapidly oxidizes ferrous to ferric iron. Nevertheless, the uptake of Fe2+ in such organisms can be studied under certain circumstances, e.g. at low ~0~. Thus, while L. delbrueckii subsp. bulgaricus (L. bulgaricus) can accumulate large amounts of iron when incubated with ferrous iron in air, at low ~02, little if any iron becomes associated with the organism. In air, hydrogen peroxide oxidizes the ferrous iron to Fe(OH)s which then binds to bacterial surface (Kot et al., 1995a). In L. acidophilus, hydrogen peroxide is not produced when glucose is present, and it is then possible to study ferrous iron uptake. The organism accumulates iron at approximately the same level as do bifidobacteria both in the presence of air and low PO:, (Kot et al., 1995b). In addition, L. acidophilus but not L. bulgaricus appears to possess a putative intracellular ferroxidase, which is also present in bifidobacteria (Kot et al., 1995a, b). L. plantarum and S. salivarius subsp. thermophilus do not elaborate significant amounts of hydrogen peroxide into the medium, yet iron accumulation is quite substantial in both (Kot et al., 1995a). It is well known that L. plantarum possesses an NADH oxidase, which produces hydrogen peroxide in the presence of oxygen. But this organism also has a peroxidase, which normally would degrade hydrogen peroxide before it had an opportunity to be extruded (Archibald, 1986). The rather substantial iron accumulation by this organism may be due to the oxidation of the internalized ferrous iron by this intracellular hydrogen peroxide before it is degraded by the peroxidase, thus maintaining very low intracelluar Fe2+ concentration. L. plantarum apparently requires manganese rather than iron for growth (Archibald, 1983). This, however, should not preclude the ability of this organism to take up ferrous iron as well, as was shown by Kot et al. (1995a). The transport of manganese in the Mn2+ form has been well studied (Archibald, 1984) and reviewed (Archibald, 1986). Since iron and manganese appear next to each other in the periodic table and their properties are therefore similar, it would be of interest to compare manganese transport in L. plantarum with iron transport in bifidobacteria. Studies with metal chelators have shown that citrate stimulates manganese uptake by the organism, and it appears as if Mn2+ is taken up as its citrate complex. In bilidobacteria, as discussed above, no carrier associated with iron transport has been identitied. Manganese uptake by L. plantarum is inhibited by DCCD, CCCP and 2,4-dinitrophenol, as is iron uptake in bilidobacteria. On the basis of such studies, the conclusion was made that ‘the proton gradient, but not ATP, is the proximal energy source required for L. plantarum Mn uptake’ (Archibald, 1986, p. 79). Because metal uptakes in bifidobacteria and L. plantarum are energy dependent, both can concentrate metals in the cell interior. Bifidobacteria can accomplish about lOO-fold concentration of iron, while L. plantarum concentrates Mn some 20&250-fold (Archibald, 1986). In both cases, such high degrees of metal
916
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accumulation are, in part, due to either oxidation or combination with a highmolecular weigh substance: in case of bifidobacteria, ferrous iron is oxidized to the insoluble Fe(OH)3, and in case of L. pluntarum, Mn2+ may be oxidized to MnOz or become bound to polyphosphate. It is clear from this brief survey that metal accumulation by Gram-positive bacterial species takes place by a number of mechanisms, some of which bear similarities to those in bifidobacteria. Each genus and species should be studied separately, as very few generalizations can be made at this point. However, it does appear that iron and other metal transport in Gram-positive organisms does not take place via the classical hydroxamate and catechol-type siderophores observed in Gram-negative bacteria and fungi. Another common denominator is that it is the divalent form of a metal that is transported into the cell interior. In Gram-negative bacteria, iron can be transported into the bacterial interior either in the ferric form as a citrate or siderophore complex, or as possibly uncomplexed ferrous iron (e.g. Crosa, 1989; Kammler et al., 1993).
FUTURE
RESEARCH
PROSPECTS
Future directions in bifidobacterial iron metabolism research point toward the identification of iron transport and storage mechanisms and the molecular entities associated therewith. Isolation of the putative divalent metal ion transporter (permease) and the intracelluar ferroxidase would be of great interest. In addition, since iron transport in bifidobacteria seems to be induced by low iron concentrations in the medium, an operon regulating this response may eventually be identified. For this purpose, the well-known fur operon associated with siderophore production and metabolism in certain Gram-negative organisms may serve as a useful model (Crosa, 1989). Iron uptake studies in bifidobacteria were carried out with micromolar concentrations of ferrous iron in the medium, and nanomolar quantities of iron were then acquired by the organism per mg dry weight. Such large quantities of iron are clearly not required for growth purposes; only a few hundred metal atoms per cell would suffice. It would be of great interest to look at ferrous iron uptake mechanisms with nanomolar quantities of iron in the medium and picomolar amounts taken up. These may prove quite different from those described above. Since electrogenic pumps appear to be involved in metal transport in Grampositive bacteria including bifidobacteria, the details of these mechanisms should be another interesting avenue of research to pursue. Current knowledge is preliminary at best, based on the effects of various inhibitors such as DCCD and CCCP. Specific porters for such metals as iron and manganese should be identified and the exact energy source established. An eventual discovery of siderophores acting as metal transporters in some Gram-positive bacteria may also be possible. The issue of clinical relevance of physiological observations on bifidobacteria must be explored. An iron-poor environment is indeed hostile to the growth of pathogens (Weinberg, 1984), but how important are bifidobacteria or even lactic acid bacteria in creating such environments in animal colons? Maximum ferrous iron uptakes have been observed in bifidobacteria and lactic acid bacteria in the
Iron metabolism
in btfidobacteria
917
presence of oxygen, yet oxygen is not present in great abundance in the colon. Would the lowered level of iron uptake, such as was observed at low ~02, still be of clinical importance with respect to maintaining the environment relatively ferrous iron-free? Of what importance is the ability of bitidobacteria to sequester iron for the preservation of various dairy products? These and other yet unresolved scientific and clinical issues provide a sound basis for further research into bifidobacterial and lactic acid bacterial physiology.
REFERENCES Alm, L. (1991). The therapeutic effects of various cultures - an overview. In Therapeutic Properties of Fermented Milks, ed. R.K. Robinson. Elsevier Applied Science, London snd New York, pp. 45-64. Archibald, F. (1983). Lactobacillus plantarum, an organism not requiring iron. FEMS Microbial. Lett., 19, 29932. Archibald, F. (1984). Manganese acquisition by Lactobacillusplantarum. J. Bact., 158, 1-8. Archibald, F. (1986). Manganese: its acquisition by and function in the lactic acid bacteria. Crit. Rev. Microbial., 13, 633109. Baker, E., Maslen, E.N., Watson, K.J. & White, A.H. (1984). Crystal and molecular structure of the ferrous ion complex of A 23187. J. Amer. Chem. Sot., 106, 28602864. Bezkorovainy, A. (1984). Iron uptake by the microaerophilic anaerobe Bifidobacterium bifidum var. pennsylvanicus. Clin. Physiol. Biochem., 2, 291-297. Bezkorovainy, A. & Solberg, L. (1989). Ferrous iron uptake by Bzjidobacterium breve. Biol. Trace Elem. Res., 20, 251-267. Bezkorovainy, A. & Topouzian, N. (1983). Aspects of iron metabolism in Btfidobacterium bifidum var. pennsylvanicus. Int. J. Biochem., 15, 361-366. Bezkorovainy, A., Miller-Catchpole, R., Poch, M. & Solberg, L. (1986a). The mechanism of ferrous iron binding by suspensions of Btjidobacterium biji’dum var. pennsylvanicus. Biochim. Biophys. Acta, 884, 6&66. Bezkorovainy, A., Topouzian, N. & Miller-Catchpole, R. (198613). Mechanism of ferrous and ferric iron uptake by Bifidobacterium biJidum var. pennsylvanicus. Clin. Physiol. Biochem., 4, 150-158. Bezkorovainy, A., Solberg, L., Poch, M. & Miller-Catchpole, R. (1987). Ferrous iron uptake by Bifidobacterium bzfidum var. pennsylvanicus: the effect of metals and metabolic inhibitors. Int. J. Biochem., 19, 517-522. Bezkorovainy, A., Solberg, L., Miller-Catchpole, R. & Poch, M. (1988). Transport of ferrous iron and lactate production in Bifidobacterium bifidum var. pennsylvanicus. Biol. Trace Elem. Res., 17, 1233137. Biavati, B., Mattarelli, P. & Crociani, F. (1992). Identification of bilidobacteria from fermented. milk products. Microbiologica, 15, 7-14. Briat, J.F. (1992). Iron assimilation and storage in prokaryotes. J. Gen. Microbial., 138, 247552483. Bullen, C.L., Tearle, P.V. & Willis, A.T. (1976). Bifidobacteria in the intestinal tract of infants: an in vivo study. J. Med. Microbial., 9, 325-334. Condon, S. (1987). Responses of lactic acid bacteria to oxygen. FEMS Microbial. Rev., 46, 2699280. Crichton, R.R. & Charloteaux-Wauters, M. (1987). Iron transport and storage. Eur. J. Biochem., 164,485-506. Crosa, J.H. (1989). Genetics and molecular biology of siderophore-mediated iron transporting bacteria. Microbial. Rev., 53, 5 177530.
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Egyed, A. & Saltman, P. (1984). Iron is maintained as Fe(I1) under aerobic conditions in erythroid cells. Biol. Trace Elem. Res., 6, 357-364. Evans, S.L., Arceneaux, J.E.L., Byers, B.R., Martin, M.S. & Aranha, H. (1986). Ferrous iron transport in Streptococcus mutans. J. Bact., 168, 10961099. Futai, M. & Tsuchiya, T. (1987). Proton transport and proton-motive force in prokaryotic cells. In Zon Transport in Prokaryotes, eds B.P. Rosen & S. Silver. Academic Press, San Diego, pp. 3-83. Guerinot, M.L. (1994). Microbial iron transport. Ann. Rev. Microbial., 48, 743-772. Hughes, D.B. & Hoover, D.G. (1991). Bilidobacteria: Their potential use in American dairy products. Food Technol., 45(4), 7483. Hughes, D.B. KcHoover, D.G. (1995). Viability and enzymatic activity of bilidobacteria in milk. J. Dairy Sci., 78, 268-276. Huschka, H.G., Muller, G. & Winkelmann, G. (1983). The membrane potential is the driving force for siderophore iron transport in fungi. FEMS Microbial. Lett., 20, 12% 129.
Ishibashi, N. & Shimamura, S. (1993). Bilidobacteria:
Research and development in Japan.
Food Technol., 47(6), 126134.
Kammler, M., Schon, C. & Hantke, K. system of Escherichia coli. J. Bact., Kot, E. & Bezkorovainy, A. (1991). depends on the metal content of its Kot, E. & Bezkorovainy, A. (1993a). Btjidobacterium
(1993). Characterization
of the ferrous iron uptake
175, 6212-6219.
Uptake of iron by Bzjidobacterium thermophilum growth medium. J. Dairy Sci., 74, 2920-2926. Effects of Mg2+ and Ca2+ on Fe’+ uptake by
thermophilum. Znt. J. Biochem., 25, 1029-1033.
Kot, E. & Bezkorovainy,
A. (1993b). Distribution
of accumulated iron in Bifidobacterium
thermophilum. J. Agri. Food Chem., 41, 177-181.
Kot, E., Miller-Catchpole, R. & Bezkorovainy, A. (1993a). Iron uptake by Btjidobacterium thermophilum protoplasts. Biol. Trace Elem. Res., 38, 1-12. Kot, E., Haloftis, G. & Bezkorovainy, A. (1993b). Ferrous iron uptake by bilidobacteria in the absence of glucose. Nutr. Res., 13, 12951303. Kot, E., Haloftis, G. & Bezkorovainy, A. (1994). Iron accumulation by bilidobacteria at low p02 and in air: action of a putative ferroxidase. J. Agri. Food Chem., 42, 685688.
Kot, E., Furmanov, S. & Bezkorovainy, A. (1995a). Ferrous iron metabolism in lactic acid bacteria and bifidobacteria. J. Food Sci., 60, 547-550. Kot, E., Furmanov, S. & Bezkorovainy, A. (1995b). Ferrous iron oxidation by Lactobacillus acidophilus and its metabolic products. J. Agri. Food Chem., 43, 1276 1282.
Kurmann, J.A. & Rasic, J.L. (1991). The health potential of products containing bilidobacteria. In Therapeutic Properties of Fermented Milks, ed. R.K. Robinson. Elsevier Applied Science, London and New York, pp. 117-l 57. Lee, Y-K. Jr Wong, S-F. (1993). Stability of lactic acid bacteria in fermented milk. In Lactic Acid Bacteria. eds S. Salminen, & A. von Wright. Marcel Dekker, New York, pp. 97-109. Neilands, J. (1981). Microbial iron compounds. Ann. Rev. Biochem., 50, 715-731. Passerat, B. & Desmaison, A-M. (1995). Lactase activity of Bifidobacterium bifidum. Nutr. Res., 15, 1287-1295. Poupard, J.A., Husain, I. & Norris, R.F. (1973). Biology of bifidobacteria. Bact. Rev., 37, 136165.
Robinson,
R.K. (1991). Micro-organisms
Fermented Milks, ed. R.K. Robinson.
of fermented milks. In Therapeutic Properties of Elsevier Applied Sciencs, London and New York,
pp. 23-43. Salamah, A.A. (1992). Effect of ethylenediamine di-0-hydroxyphenylacetic transferrin on the growth of some bacterial strains in vitro. Microbiologica, 366.
acid and 15, 361-
Iron metabolism in bifidobacteria
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Sanders, M.E. (1993). Summary of the conclusions from a concensus panel of experts on health attributes of lactic cultures: significance to fluid milk products containing cultures. J. Dairy Sci., 76, 1819-1828. Scardovi, V. (1986). Genus Bifidobacterium Orla-Jensen 1924. In Bergeys Manual of Systematic Bacteriology, Vol. 2, ed. P. Sneath. Williams and Wilkins, Baltimore, pp. 1418-1434. Schneider, W., Erni, I., Hametner, D., Magyar, B., Schwyn, B. & van Steenwijk, F. (1982). Iron (III) complexes with polyalcohols. In The Biochemistry and Physiology oflron, eds P. Saltman, and J. Hegenauer. Elsevier, New York, pp. 721-722. Topouzian, N. & Bezkorovainy, A. (1986). Iron uptake by Bifidobacterium bifidum var. pennsyIvanicus: the effect of sulfhydril reagents and metal chelators. ZRCS Med. Sci., 14, 275-276. Topouzian, N., Joseph, B.J. & Bezkorovainy, A. (1984). Effect of various metals and calcium inhibitors on the growth of Bzjidobacterium bifidum var. pensylvanicus. J. Ped. Gastroent.
Veerkamp,
Nutr., 3, 137-142.
J.H.
(1977). Effects of growth conditions on the ion composition of b$dum subsp. pennsylvanicus. Antonie Van Leeuwenhoek, 43, 11 l-l 24. E.D. (1984). Iron withholding: a defense against infection and neoplasia.
Bifidobacterium
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Physiol. Rev., 64, 64-102.
SO958-6946(96)00003-9
0
1996 Elsevier Science Limited
Printed in Ireland. All rights reserved 095%6946/96/$15.00 + 0.00
ELSEVIER
Review Article Iron Metabolism in Bifidobacteria
Anatoly Bezkorovainy, * Eva Kot, Robin Miller-Catchpole, George Haloftis & Sergey Furmanov Department
of Biochemistry, (Received
Rush Medical
15 September
College,
1995; accepted
Chicago,
IL 60612-3833,
23 December
USA
1995)
ABSTRACT Btjidobacteria are Gram-positive, anaerobic microaerophilic rods that are capable of internalizing ferrous iron at pH 5.0 and 6.5 when assayed in a post-logarithmic growth phase. Dependent upon iron concentration, iron uptake is most efficient in cells grown in a metal-depleted medium. There are two iron-uptake systems: one operating at low outside iron concentrations (1 to 20 PM),' and one operating at higher concentrations (up to 400 PM). Iron is located largely on the surface and in the cell-solublefraction at low iron concentrations. At high levels, iron is associated mostly with the insoluble (particulate) fraction. While the soluble iron is in the ferrous state, most of the insoluble iron is Fe(OH)s. It has been proposed that bijidobacteria contain an intracellular ferroxidase that oxidizes internalized Fe’+ to Fe(III) using oxygen as an electron acceptor. In B. thermophilum, the K, of this ferroxidase is 518 pM. Iron uptake by btjidobacteria depends on sugar metabolism, though minor amounts of iron are taken up in the absence of a carbon source. Iron uptake does not involve any siderophores or other carriers. The iron is apparently transported by a divalent metal permease, which requires a functioning ATPase and a proton gradient. There are many similarities between the mode of iron transport in btjidobacteria and other Gram-positive bacteria such as S. mutans and L. acidophilus, and between iron transport in btjidobacteria andmanganese transport in L. plantarum. Future work on metal transport in btjidobacteria is likely tofocus on the identification and isolation of proteins involved in iron transport and oxidation. Clinical implications of the iron uptake phenomenon in btjidobacteria are also promising areas of ,future research. Copyright 0 1996 Elsevier Science Limited *Author
to whom correspondence
should be addressed 905
906
A. Bezkorovainy et al.
INTRODUCTION Species of the genus Bifidobacterium are normally encountered in most if not all animal gastrointestinal tracts. These organisms are beneficial to humans, especially in newborns, where they maintain colonic pH at about 5 and prevent the growth of pathogens (Bullen et al., 1976). The discovery of new bifidobacterial species is an ongoing process. As of 1992, some 28 species have been identilied (Biavati et al., 1992). Though such species differ to some extent from each other morphologically and physiologically, all are Gram-positive rods with occasional bifid forms, catalase-negative, non-sporeforming, and fermenting glucose to acetate and lactate via a unique ‘bitidus’ pathway. An enzyme of this pathway, phosphofructoketolase, is used routinely to distinguish bitidobacteria from other microorganisms (Scardovi, 1986). Numerous health benefits have been ascribed to the consumption of bilidobacteria; these include alleviation of gastrointestinal infections (especially in young children), constipation and diarrhea, antitumor action, lowering of serum cholesterol and improving the immune response (Kurmann & Rasic, 1991; Alm, 1991). There has been considerable interest in determining whether or not such health claims can be rigorously confirmed (Sanders, 1993). The means for administering bifidobacteria to human beings for therapeutic or prophilactic purposes have been various dairy products, especially fermented milk and yogurt (Robinson, 1991; Hughes & Hoover, 1991; Ishibashi & Shimamura, 1993). In fact, many yogurt brands in Europe, the US and especially Japan are manufactured using bifidobacteria as one of the starter culture types. There is interest in determining whether or not bifidobacteria can significantly improve the tolerance of milk by lactose-intolerant individuals (e.g. Passerat & Desmaison, 1995), and there is concern about the relatively short periods of time that bifidobacteria can survive upon storage in various types of milk (Lee & Wong, 1993; Hughes & Hoover, 1995). Metabolism, growth characteristics and the physiological peculiarities of various bitidobacterial strains are thus intimately associated with manufacture, marketing and utilization of dairy products (Hughes & Hoover, 1991). Many beneficial effects of bifidobacteria and lactic acid bacteria in humans have been attributed to their production of organic acids (Bullen et al., 1976); however, such effects could also be the result of an efficient sequestration of iron by these bacteria, thus making it unavailable to pathogens (Bezkorovainy & Solberg, 1989). This phenomenon has often been referred to as nutritional immunity, and has usually been associated with the iron-binding properties of transferrin and lactoferrin (Weinberg, 1984). Our laboratory has been concerned with iron metabolism in bifidobacteria for a number of years. The species studied have been BiJidobacterium biJidum var. pennsyfvanicus (ATCC 11863), Bzjidobacterium breve (ATCC 15700), and Bifidobacterium thermophilum (ATCC 25866). The first two are of human origin, whereas the latter was initially isolated from bovine rumen. In our studies, we have been concerned primarily with the metabolism of ferrous iron, the form that would be available to these bacteria in their normally anaerobic habitats (Kammler et al., 1993). Very little is known about iron transport in Gram-positive prokaryotic organisms in general. Thus, review articles by Crichton & Charloteaux-Wauters (1987)
Iron metabolism in bifidobacteria
901
and by Briat (1992) make no mention of iron transport in these organisms, and that by Guerinot (1994) does so only in passing. On the other hand, iron transport in Gram-negative bacteria has been studied and documented very thoroughly and has been the subject of many reviews (e.g. Neilands, 1981; Crosa, 1989). To our knowledge, therefore, bifidobacteria appear to be the only Grampositive genus, where iron transport has been under investigation in some detail, and this has taken place in our laboratory. This review is being offered firstly as a means of calling the attention of the scientific community to this relatively uncharted area or research, and secondly, to summarize the work done in our laboratory in the past 12 years. Our objective is to present evidence that iron is a bilidobacterial growth factor, to argue in support of a ferrous iron transport hypothesis in bifidobacteria, to offer a general mechanism of such transport, and to identify the nature of accumulated iron. Comparisons between iron transport in bifidobacteria and iron and other metal transport in other Gram-positive organisms are also presented.
IRON AS AN ESSENTIAL
NUTRIENT
IN BIFIDOBACTERIA
Like most organisms, bifidobacteria require iron for growth. This was demonstrated by growing B. bifidum in the so-called modified Norris medium (Poupard et al., 1973) in the presence of various iron chelators such as EDTA and bipyridyl (predominantly ferrous iron chelators) and EDHA, 2,3_dihydroxybenzoate, desferrioxamine, 8-hydroxyquinoline and nitrilotriacetate (predominantly ferric iron chelators). The ferrous iron chelators are better growth inhibitors than the ferric ones, and such inhibition can be reversed by the addition of Fe*+ and to a lesser extent by ferric citrate (Bezkorovainy & Topouzian, 1983). Growth of bifidobacteria can be also inhibited by various divalent transition metals and this too can be relieved by the addition of Fe2+ (Topouzian et al., 1984). The mechanism for this effect is most likely based on a direct or indirect competition for a divalent metal transporter (permease) between such metals and iron. The (non)requirement of iron for the growth of lactic acid bacteria, another group of Gram-positive organisms, is more obscure, since at least one of them, Lactobacillus plantarum 14917, does not require iron for growth, but instead requires manganese (Archibald, 1983).
FERROUS
IRON UPTAKE BY BIFIDOBACTERIA
The typical iron uptake experiment involves the incubation of post-logarithmic cell suspensions (A6i0 = 1.2) with 1 to 400pM 59Fe2+ in the presence of ascorbate (as an antioxidant; Fe2+: ascorbate = 5:3), or tagged ferric citrate at pH 5.0 or 6.5 using either 0.1 M acetate buffer (pH 5.0) or 3,3_dimethylglutarate buffer (pH 5.0 and pH 6.5) in air or at low pOZ. In addition, the uptake medium, termed modified Hanks solution (mHanks; Bezkorovainy et al., 1986a), contains 0.4 g KCl, 8 g NaCl, 0.14 g CaC12 and 2 g lactose or glucose per liter. Incubations are carried out at 37°C or in an ice-water bath at t+l”C with shaking. At various times (up to 120 min), five-ml samples are withdrawn, cooled on ice and centrifuged at 4°C. The cells are then washed with ice-cold 0.1 M acetate buffer at pH
908
A. Bezkorovainy
et al.
5.0 and counted in a gamma-counter to determine the amount of iron associated with them. The quantity of cells counted represents 2.75 mg of dry weight. Lactic acid production is measured throughout the incubation (uptake) process to assess cell viability. The extent of ferrous iron accumulation by bitidobacteria depends upon their nutritional history. When grown on the regular TPY medium (Scardovi, 1986), little if any ferrous iron is accumulated. However, when exogenous metal salts are omitted, the cells take up ferrous iron readily (Fig. 1) (Kot & Bezkorovainy, 1991). A metal-poor medium, termed modified TPY (mTPY), was thus routinely used to grow bitidobacteria for iron uptake studies. A series of such experiments is illustrated in Fig. 2. It shows that lactic acid is produced as iron is accumulated, that the extent of iron accumulation depends on its concentration in the medium, and that at tM”C, there is little if any iron accumulation. Figure 3 illustrates that a carbon source is important for iron uptake, though some uptake occurs in the absence of a sugar (Bezkorovainy et al., 1986a, b; Bezkorovainy & Solberg, 1989). When cells are heated at 80°C for 15 min, the ability to accumulate iron is lost (Bezkorovainy et al., 1986a). There are three reasons why ferrous iron may become associated with bacterial cells in the above experiments: it can become oxidized by air and precipitate as Fe(OH)s, the presence of the antioxidant notwithstanding; it could become
180 160 140 120 100 80 60 40 20
100
200
300
400
Felin medun (pbll
Fig. 1. Iron accumulation by Bifidobacterium thermophilum as a function of ferrous iron concentration in the mHanks medium and its nutritional history. All incubations were carried out at 37°C for 60 min. Curve A refers to cells grown in the regular TPY medium (Scardovi, 1986); curve B refers to cells grown at [Fe’+]= 1OpM (in modified TPY medium); and curve C refers to cells grown at [Fe”] =5.5 pM (chelated modified TPY medium) (from Kot & Bezkorovainy, 1991).
Iron metabolism in bifidobacteria r
10
20
40
60
60
100
20
120
40
60
60
100
120
Time (min.)
lime (min.)
Fig. 2. Ferrous iron uptake by Bfidobacterium breve. Solid lines represent iron uptake, broken lines, lactate production. Crosses indicate &4”C, whereas circles indicate 37°C. Frame A indicates experiments with 65.1 pM Fe2+, B indicates experiments with 11.4 pM Fe’+. All experiments were performed at pH 5.0 (Bezkorovainy & Solberg, 1989).
bound to the cell surface; or it could become internalized by the cell. Air oxidation of Fe2+ can be eliminated as a possibility by performing the uptake studies at OG4”C and/or with heated cells. If some of the iron is precipitated as Fe(OH)3, it should co-centrifuge with the heated cells or with the cells used at cold
temperatures and one could then detect substantial amounts of iron associated with the cells. This does not occur. Moreover, when ferrous iron is incubated under the above conditions in the absence of bacteria and the amount of ferrous iron remaining is measured by the ferrozine reaction as a function of time, there is little if any loss of ferrous iron at both pH 5.0 and 6.5 (Bezkorovainy et al., 1988). Bifidobacteria do not produce hydrogen peroxide (Kot et al., 1995a), and lactate
u -360 -200
0" a -5 %g Et
-100
2 : J
30
60
120
Time (min.)
by B$dobacterium breve. Solid lines represent iron uptake, broken lines, lactate production. -x-x- are controls; ~ o-o are lactose-deprived cells; -o-e-- are lactose-deprived cells to which lactose was added (2 mg mL-‘) after 30 min of incubation with iron. Ferrous iron concentration was 129 pM in all experiments (from Bezkorovainy & Solberg, 1989).
Fig. 3. Effect of lactose on iron uptake and lactate production
910
A. Bezkorovainy et al.
produced by the organism, while promoting ferrous iron oxidation to some extent, sequesters the ferric iron produced and makes it unavailable for binding to the bacteria (Kot et al., 1995b). The next issue is whether or not surface binding of ferrous iron could account for iron accumulation by the cells. This can be explored by washing the iron-loaded cells with ice-cold 2 mM FeS04 and/or 2-4 mM EDTA in 0.1 M acetate buffer at pH 5.0. At this low temperature, the cellular membrane should be rigid and impermeable to any internalized iron while surface-bound iron should be washed off. Approximately 10% of the accumulated iron is normally lost by such washes (Bezkorovainy & Solberg, 1989), indicating that surface binding of ferrous iron occurs only to a minor extent. Having thus eliminated non-specific precipitation of Fe(OH)3 and surface binding of ferrous iron as major causes of ferrous iron accumulation by bifidobacteria, it becomes clear that under the conditions used in our laboratory most of the iron taken up is internalized by the cells. Further evidence of iron internalization by bitidobacteria is provided by the behavior of their protoplasts and by the effect of calimycin on the process of iron uptake. When protoplasts are prepared from iron-preloaded B. thermophilum cells, only minor losses of iron are observed, indicating that the cell wall does not serve as a major binder of iron. The protoplasts themselves cannot internalize iron, but do, instead, bind large quantities of iron on their surface. This conclusion is reached because iron accumulation by protoplasts is almost instantaneous, most if not all of the iron acquired can be eluted by 2 mM FeS04, and iron accumulation is equal at O-4” and 37°C (Kot et al., 1993a). Calimycin (A 23187) is a divalent metal ionophore, which works well with ferrous iron (Baker et al., 1984; Egyed & Saltman, 1984). When used with bifidobacteria in iron uptake experiments at concentrations of l-2 pM, iron accumulation is completely inhibited (Topouzian & Bezkorovainy, 1986; Bezkorovainy & Solberg, 1989). Such small ionophore concentrations could not bind and therefore inhibit iron uptake when the latter concentrations are 50-200 pM. Its action must therefore involve the short-circuiting of iron gradients that are formed between the cell interior and its environment. Ferrous iron accumulation by bifidobacteria takes place over a rather wide range of its concentrations (l-400 ,uM). At least two iron uptake systems are present in B. breve: the high affinity system, which operates at low iron concentrations (l-20 PM); and a low affinity system that operates at high iron concentrations (5&400 PM). The Km’s of the two systems are 35 and 86 pM, respectively (Bezkorovainy 8z Solberg, 1989). The uptake of ferric iron by bifidobacteria is very low. When ferric iron is presented to the cells as the citrate complex, little if any is accumulated. When ascorbate, a reducing agent, is added, iron accumulation resumes (Bezkorovainy, 1984). The spent bacterial growth medium, as well as the insoluble bacterial fraction obtained following sonication, are able to reduce ferric to ferrous iron using NADH as a reducing agent (Bezkorovainy et al., 1986b). This indicates the presence of a ferrireductase on the cell surface, whose function may be to bolster iron uptake when ferric but not ferrous iron is present in the growth medium. A similar situation is observed in Streptococcus mutuns (Evans et al., 1986).
911
Iron metabolism in bifidobacteria TABLE 1
Effects of Various Metal Ions and Metabolic Inhibitors on Ferrous Iron Uptake by Bilidobacteria” Group
Ion or compound
Effect
Metals
Zn2 ’ co2 + Mg2’ Ca2 ’
Inhibits Inhibits Inhibits No effect
Chelators
Bipyridyl Citrate Desferrioxamine EDHA EDTA Nitrilotriacetate
Inhibits Inhibits Inhibits Inhibits Inhibits Inhibits
lonophores
Calimycin (A23 187) CCCP Nigerisin Valinomycin Valinomycin + nigerisin
Inhibits Inhibits Stimulates Stimulates Inhibit
ATPase inhibitors
DCCD Vanadate
Inhibits Inhibits
1987); “Summarized from Bezkorovainy et a/. (1986b, Bezkorovainy & Solberg (1989); Topouzian & Bezkorovainy (1986).
THE MECHANISM
OF FERROUS IRON UPTAKE BY BIFIDOBACTERIA
The mechanism of ferrous iron uptake by bifidobacteria was investigated by using various types of metabolic inhibitors: divalent cations; metal chelators; iono hores; and enzyme/transport inhibitors (Table 1). Only one metal ion, P inhibits ferrous iron uptake competitively. The mode of action of the co* other; cannot be easily classified into any categories (Bezkorovainy et al., of 200 pM and 1987). Of special interest is Mg2+, which at concentrations higher is able to completely inhibit iron uptake by B. thermophilum. When the Mg*’ is removed from the assay medium, iron uptake resumes. Mg*’ has no effect on lactate production. Ca* + , on the other hand, has a slight stimulatory effect on iron uptake (Kot & Bezkorovainy, 1993a). It may be proposed that ferrous iron and certain other divalent cations share common permeases (transporters) on bifidobacterial plasma membranes. A number of ionophores have either a positive or a negative effect on iron uptake by bifidobacteria. The proton ionophore, CCCP, inhibits iron uptake, while valinomycin and nigericin, when used individually, stimulate iron uptake. When combined, on the other hand, valinomycin and nigericin have an inhibitory effect. ATPase inhibitors such as DCCD and vanadate have inhibitory effects
912
A. Bezkorovainy et al.
(Table 1). Since the transport of nutrients into bacterial cells depends on pH (proton) gradients and on membrane potentials (e.g. Futai & Tsuchiya, 1987), and since both can-be produced by the action of ATPases, we conclude that both the proton gradient and membrane potential are important in forcing the iron into the bifidobacterial cell interior (Bezkorovainy et al., 1987). Others, on the basis of the CCCP and DCCD data, had concluded that the proton gradient was responsible for the uptake of manganese by L. plantarum (Archibald, 1986). In the absence of glucose, when ATPase activity is minimal and protons are not being extruded from the cell interior, ferrous iron uptake is still taking place, though at drastically lowered levels. It is likely that the membrane potential is the driving force under such circumstances (Kot et al., 1993b). Membrane potential is also the driving force for siderophore-bound iron uptake in certain fungi (Huschka et al., 1983). Metal chelators are excellent iron uptake inhibitors rather than iron carriers for bifidobacteria. Such chelators can often act as iron suppliers in other bacteria, e.g. citrate for manganese (Archibald, 1984) and EDHA for iron in some Grampositive and negative organisms (Salamah, 1992). In the case of EDHA action on Gram-negative organisms, one can, in fact, postulate that siderophores are the actual iron carriers and able to retrieve iron from EDHA. The hypothesis that no classical siderophores are involved in iron transport into bifidobacteria is thus strengthened by the observed action of chelators: since siderophores have extremely high association constants for iron (Neilands, 1981) one could not expect relatively mild chelators such as citrate, bipyridyl or nitrilotriacetate to successfully compete with the carrier siderophores for iron and inhibit their action. On the other hand, these chelators are apparently able to withhold iron from the iron permease (Topouzian & Bezkorovainy, 1986). In summary, iron uptake by bitidobacteria takes place via an electrogenic pump mechanism using a permease that may be common to several divalent cations. No siderophore or other carrier is apparently involved.
’ THE NATURE
OF ACCUMULATED
IRON
Iron associated with bifidobacteria following a typical ferrous iron uptake experiment can be divided into three pools: surface-bound iron, which accounts for about 10% of total iron accumulated (see above); soluble iron in the cell cytosol; and iron associated with the insoluble cell fraction, such as the cytosol side of the plasma membrane or another intracellular structure. Intracellular distribution of iron, i.e. its partition between the soluble and insoluble pools, can be accomplished by disrupting the FeSOd-washed cells by sonication or the French pressure cell, centrifugation, and determining iron in the soluble and insoluble fractions. Further, the insoluble fraction can also be washed with 2 mM FeS04 to sub-partition that pool into elutable ferrous iron and non-elutable iron. In B. thermophi(um, the amount of iron found in the insoluble fraction increases as total cellular iron increases; at the same time, iron that is elutable by FeS04 decreases. At low cellular iron levels, most of the internalized iron can be mobilized from the cells by suspending them in iron-free buffers. As cellular iron increases, percent of mobilizable iron decreases (Bezkorovainy et al., 1988; Kot & Bezkorovainy, 1993b).
913
Iron metabolism in bifidobacteria
The question now arises as to the chemical nature of the iron pools described above. Iron in the soluble cell fraction is in the ferrous state: it is easily chelated by ion exchangers and gives a typical color with ferrozine (Kot et al., 1994). Iron bound to the particulate fraction and elutable with 2 IIIM FeS04 is most likely ferrous iron bound to membrane fragments on their cytosol side. The non-elutable particulate fraction-bound iron is most likely in the ferric form. It can be solubilized with a sorbitol-mannitol solution and such extracts absorb at 650 nm, which is typical of ferric iron complexes with polyalcohols (Schneider et al., 1982). The intracellular deposition of ferric iron following ferrous iron uptake can be further investigated by performing iron uptake experiments at low p02 (53 mm Hg). Typical results of such experiments, shown in Table 2, indicate that iron uptake by cells is higher in air than at low p02 and that the distribution of iron between the soluble and insoluble pools of the cells favors the insoluble fraction in air. Glucose amplifies this effect (Kot & Bezkorovainy, 1993b; Kot et al., 1994). The apparent intracellular oxidation of ferrous iron to the ferric form resulting in the deposition of the insoluble Fe(OH)s and its polymers in the cell can be traced to the particulate fraction, which is able to insolubilize some 50% more iron in air than at low pOz (Kot et al., 1994). It is proposed that the particulate fraction of bifidobacteria contains a ferroxidase, for which a &, of 5 18 f 130 pM with respect to Fe*+ can be calculated. This is confirmed in part when very low amounts of ferrous iron (l-l 1 PM) are used in iron uptake experiments, which are well below the Km: the low ~02 atmosphere has no effect, the ratio of insoluble to soluble cellular iron remaining the same at about 0.25 (Kot et al., 1994). We do not believe that the putative intracellular ferroxidase bears any relationship to the ferrireductase described above. The latter appears to be a dehydrogenase using NADH as a substrate, while the ferroxidase is a true oxidase using oxygen as a substrate. The ferrireductase appears to be located on the cell TABLE 2
Uptake of Iron by LMidobacteria at Low ~02 and in Air, and the Effects of Glucose. Iron Concentration
was 192 f 11.2 pM, and Incubations pH 5.0”
Microorganism
Atmosphere
B. thermophilum
Bhreve
were Carried Out at 37°C for 60 min at
Glucose (mM)
Fe2 + uptake (nmol/pellet)b
Distribution ratio’
low poz low po* air air
0.0 11.1 0.0 11.1
53.1 166 64.7 221
0.337 0.404 0.561 1.55
low po* low poz air air
0.0 11.1 0.0 11.1
41.4 135 120 312
0.268 0.243 0.844 1.67
“Adapted from Kot et al. (1994). ‘A pellet corresponds to 2.75 mg dry weight. ‘Iron in the particulate-(insoluble) fraction to that in the soluble cell fraction.
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A. Bezkorovainyet al.
surface and its mission is to generate Fe’+ from Fe(II1) for internalization purposes. The ferroxidase is intracellular and its function is apparently to remove potentially toxic quantities of Fe2+ from the cell interior. Having established that iron is internalized by bifidobacteria via an electrogenic pump mechanism and having defined the bacterial iron pools, one may now attempt to estimate the level of iron concentration by the cells with respect to their environments. Using data from Table 1 of Kot et al. (1994) we find, after correcting for surface binding of iron, that in the presence of glucose, B. breve concentrate iron some 106-fold in the presence of air and 70-fold at low p02. In the absence of glucose, the respective figures are 6%fold and 20-fold. These calculations are based on the determination that bifidobacteria have a cellular volume of 3.25 mL g-’ dry weight (Veerkamp, 1977). This concentration effect is brought about firstly by an active pumping mechanism and secondly by the fact that the internalized ferrous iron becomes oxidized to the insoluble ferric form, Proposed iron movements in the bifidobacterial cell are illustrated in Fig. 4. Note that two forms of iron are associated with the inner side of the membrane: ferrous and Fe(OH)s. It is assumed that the nutrient sugars enter the cells in symport with protons. It is also assumed that ferric iron does not enter the cell directly, but is, instead, reduced to the ferrous form first.
METAL UPTAKE BY VARIOUS GRAM-POSITIVE
BACTERIA
There is a dearth of information regarding iron and other metal transport in Gram-positive bacteria. Nevertheless, some organisms have been investigated to a sufficient extent to provide a point of reference allowing comparison with bilidobacteria. Of special interest is the metal transport in Streptococcus mutans and certain lactic acid bacteria. S. mutans has been implicated in the causation of dental decay. This organism
Glucose, lactose Glucose/Lactose
Cell membrane
+ AcetIc 8 lactic acids
Fig. 4. Proposed scheme for iron movements in a typical bifidobacterial
cell.
Iron metabolism in bifidobacteria
915
takes up iron presented in the ferric form only after reduction to the ferrous form by a membrane-bound reductase. Ferrous iron is taken up much more readily than the ferric form. As is the case with bitidobacteria, iron chelators are able to prevent the internalization of iron. Further details of iron transport in this organism have not been investigated (Evans et al., 1986). Iron metabolism in various lactic acid bacteria is more complicated than it is in bilidobacteria because many such organisms produce hydrogen peroxide in the presence of oxygen (Condon, 1987). Hydrogen peroxide rapidly oxidizes ferrous to ferric iron. Nevertheless, the uptake of Fe2+ in such organisms can be studied under certain circumstances, e.g. at low ~0~. Thus, while L. delbrueckii subsp. bulgaricus (L. bulgaricus) can accumulate large amounts of iron when incubated with ferrous iron in air, at low ~02, little if any iron becomes associated with the organism. In air, hydrogen peroxide oxidizes the ferrous iron to Fe(OH)s which then binds to bacterial surface (Kot et al., 1995a). In L. acidophilus, hydrogen peroxide is not produced when glucose is present, and it is then possible to study ferrous iron uptake. The organism accumulates iron at approximately the same level as do bifidobacteria both in the presence of air and low PO:, (Kot et al., 1995b). In addition, L. acidophilus but not L. bulgaricus appears to possess a putative intracellular ferroxidase, which is also present in bifidobacteria (Kot et al., 1995a, b). L. plantarum and S. salivarius subsp. thermophilus do not elaborate significant amounts of hydrogen peroxide into the medium, yet iron accumulation is quite substantial in both (Kot et al., 1995a). It is well known that L. plantarum possesses an NADH oxidase, which produces hydrogen peroxide in the presence of oxygen. But this organism also has a peroxidase, which normally would degrade hydrogen peroxide before it had an opportunity to be extruded (Archibald, 1986). The rather substantial iron accumulation by this organism may be due to the oxidation of the internalized ferrous iron by this intracellular hydrogen peroxide before it is degraded by the peroxidase, thus maintaining very low intracelluar Fe2+ concentration. L. plantarum apparently requires manganese rather than iron for growth (Archibald, 1983). This, however, should not preclude the ability of this organism to take up ferrous iron as well, as was shown by Kot et al. (1995a). The transport of manganese in the Mn2+ form has been well studied (Archibald, 1984) and reviewed (Archibald, 1986). Since iron and manganese appear next to each other in the periodic table and their properties are therefore similar, it would be of interest to compare manganese transport in L. plantarum with iron transport in bifidobacteria. Studies with metal chelators have shown that citrate stimulates manganese uptake by the organism, and it appears as if Mn2+ is taken up as its citrate complex. In bilidobacteria, as discussed above, no carrier associated with iron transport has been identitied. Manganese uptake by L. plantarum is inhibited by DCCD, CCCP and 2,4-dinitrophenol, as is iron uptake in bilidobacteria. On the basis of such studies, the conclusion was made that ‘the proton gradient, but not ATP, is the proximal energy source required for L. plantarum Mn uptake’ (Archibald, 1986, p. 79). Because metal uptakes in bifidobacteria and L. plantarum are energy dependent, both can concentrate metals in the cell interior. Bifidobacteria can accomplish about lOO-fold concentration of iron, while L. plantarum concentrates Mn some 20&250-fold (Archibald, 1986). In both cases, such high degrees of metal
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A. Bezkorovainy et al.
accumulation are, in part, due to either oxidation or combination with a highmolecular weigh substance: in case of bifidobacteria, ferrous iron is oxidized to the insoluble Fe(OH)3, and in case of L. pluntarum, Mn2+ may be oxidized to MnOz or become bound to polyphosphate. It is clear from this brief survey that metal accumulation by Gram-positive bacterial species takes place by a number of mechanisms, some of which bear similarities to those in bifidobacteria. Each genus and species should be studied separately, as very few generalizations can be made at this point. However, it does appear that iron and other metal transport in Gram-positive organisms does not take place via the classical hydroxamate and catechol-type siderophores observed in Gram-negative bacteria and fungi. Another common denominator is that it is the divalent form of a metal that is transported into the cell interior. In Gram-negative bacteria, iron can be transported into the bacterial interior either in the ferric form as a citrate or siderophore complex, or as possibly uncomplexed ferrous iron (e.g. Crosa, 1989; Kammler et al., 1993).
FUTURE
RESEARCH
PROSPECTS
Future directions in bifidobacterial iron metabolism research point toward the identification of iron transport and storage mechanisms and the molecular entities associated therewith. Isolation of the putative divalent metal ion transporter (permease) and the intracelluar ferroxidase would be of great interest. In addition, since iron transport in bifidobacteria seems to be induced by low iron concentrations in the medium, an operon regulating this response may eventually be identified. For this purpose, the well-known fur operon associated with siderophore production and metabolism in certain Gram-negative organisms may serve as a useful model (Crosa, 1989). Iron uptake studies in bifidobacteria were carried out with micromolar concentrations of ferrous iron in the medium, and nanomolar quantities of iron were then acquired by the organism per mg dry weight. Such large quantities of iron are clearly not required for growth purposes; only a few hundred metal atoms per cell would suffice. It would be of great interest to look at ferrous iron uptake mechanisms with nanomolar quantities of iron in the medium and picomolar amounts taken up. These may prove quite different from those described above. Since electrogenic pumps appear to be involved in metal transport in Grampositive bacteria including bifidobacteria, the details of these mechanisms should be another interesting avenue of research to pursue. Current knowledge is preliminary at best, based on the effects of various inhibitors such as DCCD and CCCP. Specific porters for such metals as iron and manganese should be identified and the exact energy source established. An eventual discovery of siderophores acting as metal transporters in some Gram-positive bacteria may also be possible. The issue of clinical relevance of physiological observations on bifidobacteria must be explored. An iron-poor environment is indeed hostile to the growth of pathogens (Weinberg, 1984), but how important are bifidobacteria or even lactic acid bacteria in creating such environments in animal colons? Maximum ferrous iron uptakes have been observed in bifidobacteria and lactic acid bacteria in the
Iron metabolism
in btfidobacteria
917
presence of oxygen, yet oxygen is not present in great abundance in the colon. Would the lowered level of iron uptake, such as was observed at low ~02, still be of clinical importance with respect to maintaining the environment relatively ferrous iron-free? Of what importance is the ability of bitidobacteria to sequester iron for the preservation of various dairy products? These and other yet unresolved scientific and clinical issues provide a sound basis for further research into bifidobacterial and lactic acid bacterial physiology.
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