JouRNAL OF BACTRIOLOGY, Apr. 1976, p. 264-271 Copyright C 1976 American Society for Microbiology
Vol. 126, No. 1 Printed in U.SA.
Role of Iron Deposition in Sphaerotilus discophorus STEPHEN R. ROGERSI* AND JAMES J. ANDERSON2
Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire 03755 Received for publication 27 October 1975
Various physiological aspects of the process of iron deposition in Sphaerotilus discophorus were examined to elucidate its role. The values of iron/protein ratios suggested that a direct relationship existed between the iron concentration of the media and the magnitude of final iron deposition. Saturation of the organism's iron deposition system occurred at a 2.0 mM iron concentration, at a value of 0.6 mg of ferric ion per mg of cell protein. Laboratory data indicated that the strain's very low capacity for iron deposition observed at low external iron concentrations makes it unlikely that it is significant in limiting iron in the natural milieu. Under optimal iron concentrations, however, strain SS1 caused precipitation of iron (adsorbed to cellular material) in broth cultures, which was 10 to 100 times that mediated by some "non-iron" microorganisms. The strain's iron requirement, which was found to be between 0.003 and 0.02 mM, is commensurate with that of other microbes. One hundred micrograms of Mn(ll) per ml and possibly 10 ug of either Co(ll) or Ni(MM) per ml could inhibit iron uptake in the deposition system. Sphaerotilus, when tested for its ability to withstand toxic concentrations of certain trace elements (Co, Cu, Mn, Ni, and Cd), demonstrated no exceptional resistance with respect to several other common microorganisms. Final cell yields were not affected by a varying iron concentration for Sphaerotilus growing under conditions of limiting carbon and nitrogen.
Sphaerotilus are often classed as "iron-precipitating bacteria" because the sheaths, which surround these filamentous organisms, can become encrusted with insoluble oxides or hydroxides of iron from their environment (4, 7). Certain Sphaerotilus strains have the concomitant ability to deposit compounds of manganese in a similar fashion (3). In a previous work (5) we developed methods for the accurate quantitation of both growth and iron deposition by this organism. It was demonstrated that a Sphaerotilus strain, exhibiting its characteristic temporal pattern of iron deposition and growth, had a growth rate that was independent of the medium iron concentration over a range of 0.02 to 4.0 mM, and that there was no evidence delineating a direct relationship between this iron concentration and final cell protein yields. Furthermore, it was concluded that protein synthesis is not required by this organism at the time during which the major portion of iron precipitation occurs. We suggested that the data were consistent with a hypothesis implicating certain of the constituents of the
organism's sheath as important in mediating iron deposition. This work, however, did not elucidate any specific role for the process of iron deposition in Sphaerotilus. Furthermore, characterization of this phenomenon is pertinent in view of investigations which suggested that autotrophic use of iron and/or manganese by this organism is unlikely (2, 9). The problem was approached by testing several hypotheses: (i) that the organism's capacity to remove iron might starve other competing organisms with high iron requirements, (ii) that iron deposition is useful in fulfilling the organism's physiological iron requirement under conditions of low exogenous iron, (iii) that iron deposition serves a protective function which might facilitate resistance to toxic concentrations of iron, manganese, or other chemically similar trace elements. The following work was designed to explore these hypotheses, as well as to investigate several other points pertaining to the possible role of the iron deposition process in S. discophorus. MATERIALS AND METHODS Bacterial strains. S. discophorus strain SS1, a manganese oxidizing strain, has been previously described (5). S. discophorus strain Vl was derived from a parent similar to strain SS1 by heat shock.
' Present address: Sloan-Kettering Institute for Cancer Research, Donald S. Walker Laboratories, Rye, N.Y. 10580. 2 Present address: Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Mich. 48103.
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ROLE OF IRON DEPOSITION IN SPHAEROTILUS
Strain Vi is similar to strain SS1, except for the inability to oxidize manganous salts and a negligible capacity for iron deposition. It appears to be analogous to the "S strains" of S. discophorus isolated by Stokes and Powers (8). The isolation and purification of S. discophorus strain SS1 is described elsewhere by Rogers and Anderson (5). Strain Vi was isolated from a parent strain similar to SS1 after a heat shock, consisting of exposure to a temperature of 45 to 50 C for approximately 30 min. The treatment produced a variant strain which had lost the ability to oxidize manganese. The capability was not regained after more than one year of semi-monthly transfers. The three nonfilamentous bacterial strains used in the comparative experiments are Escherichia coli strain B/r, Aerobacter aerogenes strain 1033, and Pseudomonas denitrificans strain DMS-6. The latter two strains were obtained from the culture collection of the Dartmouth Medical School. Media. The basal medium employed in the experiments, as well as the stock culture medium, has been described previously (5). The basal medium contained: peptone (Difco), 0.15%; yeast extract (Difco), 0.10%; N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid (HEPES) from Calbiochem, 10 mM; ammonium citrate, 0.028%; MgSo4 7H20, 0.02%; and CaCl2, 0.005% in tap water at pH 7.1. Iron was added as FeCl3 6H20 where indicated. Medium "Ml" designates broth medium which contained the basal medium, modified as indicated previously, with radioactive iron (as 59Fe ferric chloride to achieve 0.01 MCi/ml) and/or L[3H]alanine (specific activity 13.2 Ci/mmol added at a level of 0.1 ,uCi/ml) from the New England Nuclear Corp. Growth and harvesting of cells. All cultures were incubated at 18 C in a humidified atmosphere without agitation. Inoculation was done by adding a suspension, which consisted of unlabeled exponential-phase cells grown in 0.02 mM iron, to produce an initial cell density of 105/ml. For the "dilute media" experiments, the cells were centrifuged (4 min at 1000 x g), washed, recentrifuged, and suspended in a buffer-salts solution (basal medium without peptone and yeast extract). Samples (1 ml) of all cultures were removed, treated with a 5% (vol/vol) trichloroacetic acid solution (containing 20 mM sodium citrate), filtered, and washed as described previously (5). Radioactive amino acid and iron uptake assays. Each sample, contained on a dried glass fiber filter disk, was prepared for liquid scintillation counting as described by Rogers and Anderson (5). The counting was done by using a Packard Instruments liquid scintillation spectrometer, which was adjusted to discriminate and record 3H and 59Fe emissions simultaneously. All samples were quench corrected and converted to estimates of cell protein and iron (incorporation per milliliter of original culture), which has also been previously described (5). Quantitative analyses. Mathematical manipulation of data (quench correction and experimental variation statistics) were done by using computer programs which were compiled and run on the Dart-
265
mouth Time Sharing System. The experimental variation which is indicated, where significant, on all plots and tables represents plus and minus one standard error of the mean (n 2 3). RESULTS
Quantitative evaluation of iron deposition. The previous data concerning the characterization of the growth and iron deposition processes (5) presented iron accumulation as counts (59Fe
dpm) or micrograms of iron per milliliter of culture medium. Because fluctuations in cell populations will also affect the total amount of iron deposited, the data can be normalized and presented as iron/protein ratios. This correction allows the comparison of iron deposition over a parametric iron concentration range. A summary of growth and iron deposition is presented, as mean final iron/protein ratios (after ca. 120 h), with the associated iron concentrations in the left portion of Table 1. The lack of influence of a varying iron concentration upon growth rates and final cell yields has been noted previously (5). The data show a definite increase in the quantity of deposited iron as its concentration is increased in the medium. The ratio increases and reaches a maximum as the iron concentration in the medium is increased from 0.02 to 4.0 mM. It appears that the ability for iron deposition becomes saturated in these laboratory cultures only at an extremely high iron concentration of approximately 2.0 mM. The saturation is alternatively demonstrated by the reciprocals of both the final iron/protein ratios and the associated iron concentrations (right portion of Table 1), which are linearly related (regression coefficient significant at P s 0.001).
Growth in low iron. Because Sphaerotilus seemed to elicit the same growth responses to a wide variety of iron concentrations, no obvious role for the process could be inferred. It seemed possible, however, that this capability TABLE 1. Sphaerotilus, strain SS1, mean final iron/ protein ratios, the associated iron concentrations, and their reciprocals Fe (mM)
0.02 0.10 0.50 1.0 2.0 4.0
Ratio,
0.018 0.17 7.7 15 58 60.5
1/Fe (1/mM)
50 10 2 1 0.5
0.25
1/Ratio°
55.6 5.9 0.13 0.067 0.017 0.0166
Fe/protein (microgram per microgram x 100) after 120 h of incubation. b Protein/Fe (micrograms of Fe/microgram of protein x 100)-'. a
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ROGERS AND ANDERSON
might b4e used to insure adequate amounts of metaboliic iron. To establish the threshold of the iron reqiuirement of strain SS1, cultures were incubate d in medium Ml with no supplemental iron. Thie resulting mediuin, containing approximaltely 0.003 mM iron, did not support growth atfter inoculation and incubation for 136 h. Figurfe 1 is a plot of the growth of two parallel cultuires of equal inocula and incubation conditioras. The 1 mM control curve is derived from a c-ulture containing labeled amino acid and 1 m! M labeled iron from the time of inoculation. Th; at curve representing an iron addition was deri ived from a culture initially containing only L-[3 'H]alanine. The basal iron level of this medium , as with the previous experiment, was approxinnately 0.003 mM. At 54 h after inoculation (arrow), labeled iron was aseptically added to the cl alture, bringing the final iron concentration t;o 1.0 mM, the control level. Growth in the cont rol is similar to previous experiments. The low iron culture displays, at most, a very depresse,d growth until the iron level in the medium is increased. An immediate exponential grovvth period is then observed. Both rates , , , w ' looc)
boc
C
Q) 0 ._c
I
IO FIG.
1
130 90 HOURS .Growth of Sphaerotilus strain SSJ with 50
limiting iron. Control curve represents protein in medium Ml containing labeled 1 mM total iron con-
J. BACTERIOL.
and final yields correspond to those of the control but are delayed by the time preceding the iron addition. Iron deposition in the control and experimental cultures (after iron addition) were both consistent with earlier data. The fact that the organisms fail to grow until after the 0.02 mM iron supplement suggests that the strain's iron requirement is greater than 0.003 mM but not more than 0.02 mM, quite within that range described generally for other gram-negative bacteria (10). It would appear that the capability of iron deposition is of no particular advantage to this organism in sequestering metabolic iron. Trace metals' effect upon final growth and iron deposition. In view of the ability of some Sphaerotilus strains to deposit oxides of manganese as well as iron, the effects of various other trace metals upon the final growth and iron deposition process of strain SS1 were tested. It was thought that this mechanism(s) might provide increased tolerance to toxic concentrations of other trace elements. Five trace metal ions, Co(II), Cu(II), Cr(III), Mn(II), and Ni(II), were chosen because of their proximity to iron in the periodic table (chemical similarity), their essentiality to microorganisms, and their acknowledged toxicity at moderate concentrations (1). Each metal was added to medium Ml containing 1.1 ,ug of iron per ml (0.02 mM) in five 10-fold serial dilutions from 100 to 0.01 ,ug/ ml. The effects of these metals on growth and iron deposition, as percentages of final protein and iron deposited in the control culture (containing only iron), are compared in Table 2 (after 120 h). A general toxic effect at 100 ug/ml can be observed as a marked inhibition of growth in all but the Mn(II) cultures. For the five lower concentrations (0.01 to 10 ,ug/ml), no appreciable inhibition can be demonstrated to result from adding copper, chromium, or manganese. Partial reduction in accumulated cell protein, along with a disproportionately greater decrease in iron deposition, is found with manganese at 100 ,ug/ml, suggesting a competition with iron. However, this competition was very weak, because the manganese was 100 times more concentrated than the iron. Weak competition may also occur with cobalt and nickel at the 10-gg/ml level. No inhibitory couldor be demonstrated effect of any metal to 1 ug/ml. at equal concentrations less than As a basis ffor evaluation, some qualitative . r comparsons of the growth of strains of A. aerogenes, E. coli, and P. denitrificans, in the same medium, supplemented with 1, 10, or 100 ,ug of the trace metals per ml, are provided in Table 3. It is obvious that A. aerogenes and P. denitri-
the outset. The delayed addicell protein (0) of cultures grown in medium Ml, initially containing no labeled supplemE ental iron (basal level of 0.003 mM), and ficans, under these conditions, exhibit a high then labe?led at 54 h (arrow) with 1 mM total iron. tinuousl3 y present from tion curtve represents
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ROLE OF IRON DEPOSITION IN SPHAEROTILUS
267
TABLE 2. Growth and iron deposits of Sphaerotilus strain SSI in medium Ml containing various concentrations of trace metals after 120 h of incubation Ion additiona
Ion concn Protein + % erroe (zg/Ml) (~g/m1)(yg/ml) (~±gIml)
None Co(II)
119 ± 3.8 0.01 112 ± 1 0.10 113 ± 4.4 (1 Lg/ml = 0.017 mM) 1.0 115 ± 3.1 10 77.8 + 3.7 100 NG' CU(II) 0.01 104 ± 2.2 0.10 110 ± 2.0 (1 i.g/ml = 0.016 mM) 1.0 114 ± 4.9 10 129 + 3.4 100 NG Cr(III) 0.01 122 t 2.4 0.10 112 ± 3.9 (1 yg/ml = 0.019 mM) 1.0 112 ± 4.6 10 118 ± 4.6 100 NG Mn(II) 0.01 100 ± 0.9 0.10 108 + 3.2 (1 AgIml = 0.018 mM) 1.0 119 ± 8.5 10 126 ± 1.5 100 86 ± 5.3 Ni(II) 0.01 118 ± 4.0 0.10 109 ± 1.0 (1 jg/ml = 0.017 mM) 1.0 114 ± 3.2 10 79 ± 12.7 100 NG aAll cultures contained 0.02 mM FeC13. b Derived from the standard error of the mean. 'NG, No growth at this metal concentration. ' ND, No iron deposition at this concentration.
tolerance to the metals, growing in every case in concentrations of up to 10 ,ug/ml. All three genera grew at 100 ug of manganese per ml and P. denitrificans exhibited good growth even with 100 ,tg of copper added per ml. E. coli appeared to be somewhat more sensitive, showing inhibition at 10 lug of cobalt and copper per ml. S. discophorus, therefore, fail to demonstrate any exceptional tolerance to the trace elements tested with respect to several other common bacteria. Although the laboratory strains examined may themselves possess above average tolerance to the metals tested, the fact that they do not deposit iron or manganese, as does S.discophorus, does not support the hypothesis that iron deposition in this organism plays an exclusive role in toxic element tolerance. Sphaerotilus could conceivably compete with these genera in a natural mixed microbial population and, as far as the laboratory data indicate, would have no apparent advantage in this respect. Furthermore, the lack of strongly disproportionate effects of all elements, except manganese, upon iron deposition
Percentage of control
protein 100 94.2 95.2 93.3 65.4
87.7 92.7 96.0 109 103 94.5 94.5 99.4 84.4 90.8 99.9 106 72.2 98.7 91.2 95.5 66.9
Fe ± % error
Percentage
of control OAglml) (.gm)Fe
0.146 ± 0.155 ± 0.147 ± 0.167 + 0.066 ± NDd 0.155 ± 0.172 t 0.135 ± 0.167 ± ND 0.174 ± 0.155 ± 0.155 ± 0.155 ± ND 0.159 ± 0.165 + 0.135 ± 0.142 ± 0.0056 ± 0.162 ± 0.176 ± 0.152 + 0.061 ± ND
2.5 7.3 8.9 7.2 3.0
100 106 101 115 45.3
7.8 2.6 16.2 7.0
107 118 92.7 115
3.0 1.8 9.6 2.4
119 107 106 107
5.2 0.3 6.1 4.8 12.4 10 5.2 8.7 83
109 113 93.2 97.6 3.9 112 120 105 41.8
suggests that they do not participate or compete with iron, which has a relatively specific precipitation mechanism. Comparison of iron deposition in selected genera. To establish the relative ability of strain SS1 to precipitate (adsorb) iron, deposition among S. discophorus strain SS1, P. denitrificans, and strain Vl (a S. discophorus strain incapable of manganese oxidation) is compared in Table 4. Iron/protein ratios are used to normalize the differences which might have occurred in final cell mass per milliliter of culture. The ratio for P. denitrificans remains fairly constant during the incubation. Strain Vl exhibits a very low ratio until the final growth point, at which time it attains approximately the same range as that of P. denitrificans. S. discophorus strain SS1 shows its normal high ratio, with final values ranging from 15- to 40-fold greater than those obtained from the strain Vl and P. denitrificans cultures. Because the latter organism produced a pH increase from 7.1 to 7.7 during the incubation period, it is possible that the iron deposition noted in this case can be partly attributed to a
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ROGERS AND ANDERSON
J. BACTERIOL.
TABLE 3. Comparison ofgrowth with added trace metals of different genera Mn (III) Cr (III) Cu (II) Co (II) Strain
1.0" 10 ++b ++
1.0
10
100
1.0
10
100
1.0
10
100
-C ++ Aerobacter aerogenes +d + Escherichia coli ++ Pseudomonas denitrificans ++ ++ a Micrograms per milliliter. b Culture very turbid after 50 h of incubation. c No visible turbidity. d Visible turbidity. eCr(III) caused precipitate at 100 jig/ml.
+ ++
++
++ + ++
++ + ++
*e * *
+ + ++
+ + ++
+ + ++
100
TABLE 4. Comparison of Fe/protein ratios of Sphaerotilus strains SS1 and Vi and P. denitrificans strain DMS-6 Fe/protein ratioa
Strain 39.5 h
48.25 h
64 h
71 h
89.5 h
111.5 hb
5.2 ±8.4 0.036 ± 16.7
5.6 ±4.4 0.065 ± 27.1
7.5 ±4.9 0.42 ± 39.9
17.25 h
41.75 h 48.75 hb
26 h
Sphaerotilus SS1 Vi
0.13
0.43
3.8
+34b
±42.9
±11.4
0.024 ± 71
0.036 ± 38.9
P. denitrificans DMS-6 a Fe/protein ratios given as (micrograms/disintegration per minute) of the mean (percent). b Stationary-phase cells.
purely chemical process. The pH fluctuation has, however, been rigorously examined for strain SS1; the contribution ofthis phenomenon to iron deposition during its growth is negligible. The loss of manganese deposition in strain Vl parallels the loss of iron deposition down to the level of an ordinary (non-iron) pseudomonad. This in turn is strongly suggestive of common causes in the deposition of both ions. Growth at different iron concentrations in dilute media. The use of a "rich" medium for growth of Sphaerotilus may not be an accurate indication of its behavior vis A vis the physiological significance of iron deposition. We questioned, therefore, whether a changing iron concentration could measurably affect final growth of Sphaerotilus in medium containing more dilute organic components, approximating the natural habitat. The effect of successive dilution ofthe organic components (peptone and yeast extract only) of medium Ml upon the final growth yield of Sphaerotilus strain SS1 is shown in Fig. 2. A successive decline in total cell mass with increasing dilution of the carbon and nitrogen source is as expected. To test any effect that a varying iron concentration might have upon final cell yields at the resulting very low growth rates, several diltutions were chosen
x
0.20 0.16 0.13 0.16 ±47.4 ± 4.3 ±12.9 ± 1.8 104 ± variations as standard errors
which produced low, but reliably measurable, growth. The cultures were then incubated in iron concentrations of 0.02, 0.10, and 0.50 mM 120 1-%
80 t I-
.c )
40 H
10
20
30
(Organic Dilution)FIG. 2. Growth of Sphaerotilus strain SS1 in dilute media. Final culture cell protein was estimated from -[3H]alanine incorporation (see Materials and Methods) and plotted versus the reciprocal of the dilution of the organic component (peptone and yeast extract) of medium Ml. Error bars represent the standard error of the mean.
ROLE OF IRON DEPOSITION IN SPHAEROTILUS
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269
TABLE 5. Cellular growth yields of Sphaerotilus strain SSZ as a function of nutrient and Fe(III) dilutions p.g of protein/ml of culture Dilution of orPredicted' ganic componenta
0.02 mM Fe
0.10 mM Fe
0.5 mM Fe
100 103 ± 2.2 94 ± 3.9 20 40 ± 6.8 54 ± 9.4 10 24 ± 14.7 19 2 4 11 ± 14.1 10 ± 10. ld 41.6d 2 4 ± 4.4 6 11.3 57.2 1 0.7 ± 60.7 0.8 ± 39.9 48.5 a (Medium peptone + yeast extract)/(medium Ml peptone + yeast extract). b Assuming that cells yields are simply and directly proportional to concentrations of nutrients regardless of iron concentration. c Second value equals percentage. d Difference statistically significant at the P = 0.05 level. 0 0.20 0.10 0.04 0.02 0.01
93± 44± 17 ± 5 ± 0.5 ± 0.7 ±
5.6"
4.1 3.7
until maximum cell growth was achieved. The resulting cell yields are compared in Table 5. The table lists yields at each of the three different iron concentrations as the organic components of medium Ml are diluted between 0.20 and 0.01 times their normal concentrations. These data represent the results of two different experiments. The individual values comprise a minimum of three and a maximum of six individually inoculated _[3H]alanine-labeled cultures. Pairwise comparisons were made by using the "t" and "F"' tests and the Student-Newmans-Keuls tests on pooled data (0 and 0.04 dilutions). They indicated that, within each dilution, the only (marginally) significant difference (P = 0.05) occurs at the 0.04 dilution, between the 0.02 and 0.10 mM iron cultures. It can therefore be concluded that these data do not support the hypothesis of the proposed differential effect of iron concentration on cell yields for populations growing in rich versus dilute media. DISCUSSION The question posed throughout the investigation concerned the role of the iron deposition process in this organism. The iron/protein ratios, derived from various experiments, indicate the existence of a direct relationship between the extent of iron deposition and the iron concentration in the medium up to a saturation point. The organism's iron deposition capability became saturated only at a high iron concentration of approximately 2 mM. Parenthetically, these saturation data measured a maximum level of iron removal by Sphaerotilus in laboratory culture, which suggests that it cannot exercise significant control over the iron cycle in the natural milieu. Cells growing in a 0.02 mM iron concentration possess this capacity (0.6 mg of iron per mg of cell protein), yet they extracted only about 1.8 x 10-4 mg of iron per mg
of cell protein. The equilibrium, therefore, already favored dissolved iron at this level. A limiting role played by iron-precipitating bacteria in the natural iron cycle (6) is highly unlikely. It was proposed that iron deposition may represent a kind of iron detoxification mechanism for Sphaerotilus. Although far from removing significant quantities of this element, iron deposition could provide protection either by changing the microenvironment around the cell, by rendering the cell and/or its surrounding sheath less permeable to iron, or by preventing access of the toxic metal to the cell membrane. These processes could increase the organism's tolerance to high concentrations of the element. The hypothesis that iron deposition per se is required for a protective function is not supported, because other species of noniron-depositing bacteria (P. denitrificans and E. coli) grew well at 0.5 mM concentrations of this moderately toxic (1) element. The iron requirement of S. discophorus strain SS1 was defined by experiments measuring growth at low iron concentrations. We established the minimal requirement as being in the 0.003 to 0.02 mM range. This is similar to the requirement of E. coli growing on acetate (Neidhardt, personal communication) and suggests that the iron deposition mechanism is not particularly advantageous to this organism in satisfying the requirement. In the comparisons of iron deposition capability, Sphaerotilus strain SS1 demonstrates quantitatively the well-known distinction of the iron-depositing bacteria from other bacteria by its ability, in the laboratory, to attain iron/ protein ratios between 10- and 100-fold greater than those observed in S. discophorus strain Vl and in P. denitrificans cultures. It is probable that S. discophorus strain Vl (nonmanganese-depositing strain), as evidenced by its
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ROGERS AND ANDERSON
observed morphological and physiological characteristics, is identical to the S strains of S. discophorus, which were isolated by Stokes and Powers (8). These strains were identical to the wild-type strains, with the exception that the S strains lacked the ability for sheath production and for manganous salt oxidation. Microscopy examinations suggested that sheath production by S. discophorus strain Vi, compared to strain SS1, is absent, or at least minimal. Strain Vi demonstrated a reduced capacity to deposit iron, which was commensurate with that of P. denitrificans, a non-iron bacterium, although it could still grow normally in relatively high iron (1.5 mM). This evidence supports the proposed link between sheath production and manganese and iron deposition and, furthermore, does not confirm the hypothesis of iron tolerance conferred by the process. A criticism could be leveled at the extrapolation of the findings of a laboratory culturing system to the organism's behavior in the environment. Results obtained with dilute media demonstrate that the use of the in vitro system probably does not create misleading information concerning iron concentration and its inability to affect the yield of S. discophorus cells. When the organism's growth rate was significantly decreased by the nutrient limitation imposed by diluting the peptone and yeast extract components of the media, no significant differences in the final cell yields could be demonstrated at iron concentrations between 0.02 and 0.50 mM. Metal deposition in Sphaerotilus was hypothesized as being a detoxification mechanism, which might provide the bacterium with a resistance to growth inhibition caused by high concentrations of certain trace elements. We questioned whether other elements, with chemical properties similar to those of iron, could interfere with the process used for iron deposition, and whether S. discophorus strain SS1 was unusually resistant to the toxic effects of these ions. Salts of cobalt, copper, chromium, manganese, and nickel were added to the cul.. ture medium so that their effect upon iron deposition and growth could be observed. Athough SS1 did show a tolerance to these metals at concentrations up to 10 ,ug/ml, the comparison to other genera indicated nothing exceptional about the response. The results indicated that strain SS1 is of equal or greater susceptibility to growth inhibition by these metals, compared to other selected microbes. Sphaerotilus growth and iron deposition was also observed in the presence of manganous ions. The strain comparison indicated that a 100-,ug concentration of
J. BACTERIOL.
manganese per ml is not inhibitory to the other microbes which were compared to Sphaerotilus. The disproportionate decrease in iron deposition of Sphaerotilus (to 3.9% of the control), accompanied by growth that was depressed to 72.2% of the control, indicated preferential inhibition of iron uptake, although selectivity for iron is apparent by the magnitude of Mn(II) necessary to achieve this inhibition. The competitive effect was achieved only after manganese was supplied in quantities which were almost 100-fold greater than those of iron (0.02 mM iron and 1.82 mM manganese). This result is not consistent with a deposition mechanism for which the principle substrate is manganese; therefore, iron precipitation is not a simple byproduct of manganese oxidation. Oxidation of manganese, however, might be favored in Sphaerotilus growing under certain conditions of low oxygen tension in the presence of sulfide. It is possible that the same phenomenon may occur when either Co(II) or Ni(II) is added (10 ,ug/ml or approximately 0.2 mM), although the disproportional decreases of iron deposition and growths were less pronounced. Reciprocal experiments (iron inhibition of labeled manganese oxidation) are necessary to establish the duality of the mechanism. S strains of S. discophorus, which arose by an unknown consequence of heat shock (see Materials and Methods), apparently have lost both iron and manganese deposition capabilities. This fact suggests relationships between the two, but until the basis for the conversion is known it would be premature to ascribe both losses to a simple genetic (mutation?) event. The fact that S strains have identical growth rates and yields as their parent again illustrates the lack of a gross physiological role for ion deposition. Mulder (2) and van Veen (9) proposed that manganese oxidation was protein facilitated, and that this process exhibited a behavior which was characteristic of an enzyme-catalyzed reaction. The present investigation suggests that iron deposition and manganese oxidation might share the same mechanism. We speculate that a moiety of the organism's sheath is responsible for catalyzing the deposition of both manganese and iron, although the specific function of this phenomenon is elusive. ACKNOWLEDGMENTS Financial support for this work was provided in part by the Cramer Foundation of Dartmouth College and by Sigma Xi Grants-in-Aid for Research. LITERATURE CITED 1. Bowen, H. J. M. 1966. Trace elements in biochemistry. Academic Press Inc., New York.
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ROLE OF IRON DEPOSITION IN SPHAEROTILUS
2. Mulder, E. G. 1972. Le cycle biologique et aquatique du der et du manganese. Rev. Ecol. Biol. Sol. 9:321-348. 3. Mulder, E. G., and W. L. van Veen. 1963. Investigation
the Sphaerotilus-Leptothrix group. Antonie van Leeuwenhoek J. Microbiol. Serol. 29:121-153. 4. Pringsheim, E. G. 1949. The filamentous bacteria Sphaerotilus, Leptothrix, and Cladothrix, and their relation to iron and manganese. Phil. Trans. R. Soc. London Ser. B 233:453-482. 5. Rogers, S. R., and J. J. Anderson. 1976. Measurement of growth and iron deposition in Sphaerotilus discophorus. J. Bacteriol. 126:257-263. 6. Ruttner, F. 1952. Iron and manganese, p. 83-87. In D. on
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G. Frey and F. E. J. Fry (ed.), Fundamentals of limnology. University of Toronto Press, Toronto. 7. Starkey, R. L. 1945. Transformations of iron by bacteria in water. J. Am. Water Works Assoc. 37:963-984. 8. Stokes, J. L., and M. T. Powers. 1965. Formation of rough and smooth strains of Sphaerotilus discophorus. Antonie van Leeuwenhoek J. Microbiol. Serol. 31:157-164. van Veen, W. manganese
L. 1972. Factors affecting the oxidation of by Sphaerotilus discophorus. Antonie van Leeuwenhoek J. Microbiol. Serol. 38:623-626. 10. Weinberg, E. D. 1974. Iron and susceptibility to infectious disease. Science 194:952-956. 9.
Vol. 126, No. 1 Printed in U.SA.
Role of Iron Deposition in Sphaerotilus discophorus STEPHEN R. ROGERSI* AND JAMES J. ANDERSON2
Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire 03755 Received for publication 27 October 1975
Various physiological aspects of the process of iron deposition in Sphaerotilus discophorus were examined to elucidate its role. The values of iron/protein ratios suggested that a direct relationship existed between the iron concentration of the media and the magnitude of final iron deposition. Saturation of the organism's iron deposition system occurred at a 2.0 mM iron concentration, at a value of 0.6 mg of ferric ion per mg of cell protein. Laboratory data indicated that the strain's very low capacity for iron deposition observed at low external iron concentrations makes it unlikely that it is significant in limiting iron in the natural milieu. Under optimal iron concentrations, however, strain SS1 caused precipitation of iron (adsorbed to cellular material) in broth cultures, which was 10 to 100 times that mediated by some "non-iron" microorganisms. The strain's iron requirement, which was found to be between 0.003 and 0.02 mM, is commensurate with that of other microbes. One hundred micrograms of Mn(ll) per ml and possibly 10 ug of either Co(ll) or Ni(MM) per ml could inhibit iron uptake in the deposition system. Sphaerotilus, when tested for its ability to withstand toxic concentrations of certain trace elements (Co, Cu, Mn, Ni, and Cd), demonstrated no exceptional resistance with respect to several other common microorganisms. Final cell yields were not affected by a varying iron concentration for Sphaerotilus growing under conditions of limiting carbon and nitrogen.
Sphaerotilus are often classed as "iron-precipitating bacteria" because the sheaths, which surround these filamentous organisms, can become encrusted with insoluble oxides or hydroxides of iron from their environment (4, 7). Certain Sphaerotilus strains have the concomitant ability to deposit compounds of manganese in a similar fashion (3). In a previous work (5) we developed methods for the accurate quantitation of both growth and iron deposition by this organism. It was demonstrated that a Sphaerotilus strain, exhibiting its characteristic temporal pattern of iron deposition and growth, had a growth rate that was independent of the medium iron concentration over a range of 0.02 to 4.0 mM, and that there was no evidence delineating a direct relationship between this iron concentration and final cell protein yields. Furthermore, it was concluded that protein synthesis is not required by this organism at the time during which the major portion of iron precipitation occurs. We suggested that the data were consistent with a hypothesis implicating certain of the constituents of the
organism's sheath as important in mediating iron deposition. This work, however, did not elucidate any specific role for the process of iron deposition in Sphaerotilus. Furthermore, characterization of this phenomenon is pertinent in view of investigations which suggested that autotrophic use of iron and/or manganese by this organism is unlikely (2, 9). The problem was approached by testing several hypotheses: (i) that the organism's capacity to remove iron might starve other competing organisms with high iron requirements, (ii) that iron deposition is useful in fulfilling the organism's physiological iron requirement under conditions of low exogenous iron, (iii) that iron deposition serves a protective function which might facilitate resistance to toxic concentrations of iron, manganese, or other chemically similar trace elements. The following work was designed to explore these hypotheses, as well as to investigate several other points pertaining to the possible role of the iron deposition process in S. discophorus. MATERIALS AND METHODS Bacterial strains. S. discophorus strain SS1, a manganese oxidizing strain, has been previously described (5). S. discophorus strain Vl was derived from a parent similar to strain SS1 by heat shock.
' Present address: Sloan-Kettering Institute for Cancer Research, Donald S. Walker Laboratories, Rye, N.Y. 10580. 2 Present address: Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Mich. 48103.
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Strain Vi is similar to strain SS1, except for the inability to oxidize manganous salts and a negligible capacity for iron deposition. It appears to be analogous to the "S strains" of S. discophorus isolated by Stokes and Powers (8). The isolation and purification of S. discophorus strain SS1 is described elsewhere by Rogers and Anderson (5). Strain Vi was isolated from a parent strain similar to SS1 after a heat shock, consisting of exposure to a temperature of 45 to 50 C for approximately 30 min. The treatment produced a variant strain which had lost the ability to oxidize manganese. The capability was not regained after more than one year of semi-monthly transfers. The three nonfilamentous bacterial strains used in the comparative experiments are Escherichia coli strain B/r, Aerobacter aerogenes strain 1033, and Pseudomonas denitrificans strain DMS-6. The latter two strains were obtained from the culture collection of the Dartmouth Medical School. Media. The basal medium employed in the experiments, as well as the stock culture medium, has been described previously (5). The basal medium contained: peptone (Difco), 0.15%; yeast extract (Difco), 0.10%; N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid (HEPES) from Calbiochem, 10 mM; ammonium citrate, 0.028%; MgSo4 7H20, 0.02%; and CaCl2, 0.005% in tap water at pH 7.1. Iron was added as FeCl3 6H20 where indicated. Medium "Ml" designates broth medium which contained the basal medium, modified as indicated previously, with radioactive iron (as 59Fe ferric chloride to achieve 0.01 MCi/ml) and/or L[3H]alanine (specific activity 13.2 Ci/mmol added at a level of 0.1 ,uCi/ml) from the New England Nuclear Corp. Growth and harvesting of cells. All cultures were incubated at 18 C in a humidified atmosphere without agitation. Inoculation was done by adding a suspension, which consisted of unlabeled exponential-phase cells grown in 0.02 mM iron, to produce an initial cell density of 105/ml. For the "dilute media" experiments, the cells were centrifuged (4 min at 1000 x g), washed, recentrifuged, and suspended in a buffer-salts solution (basal medium without peptone and yeast extract). Samples (1 ml) of all cultures were removed, treated with a 5% (vol/vol) trichloroacetic acid solution (containing 20 mM sodium citrate), filtered, and washed as described previously (5). Radioactive amino acid and iron uptake assays. Each sample, contained on a dried glass fiber filter disk, was prepared for liquid scintillation counting as described by Rogers and Anderson (5). The counting was done by using a Packard Instruments liquid scintillation spectrometer, which was adjusted to discriminate and record 3H and 59Fe emissions simultaneously. All samples were quench corrected and converted to estimates of cell protein and iron (incorporation per milliliter of original culture), which has also been previously described (5). Quantitative analyses. Mathematical manipulation of data (quench correction and experimental variation statistics) were done by using computer programs which were compiled and run on the Dart-
265
mouth Time Sharing System. The experimental variation which is indicated, where significant, on all plots and tables represents plus and minus one standard error of the mean (n 2 3). RESULTS
Quantitative evaluation of iron deposition. The previous data concerning the characterization of the growth and iron deposition processes (5) presented iron accumulation as counts (59Fe
dpm) or micrograms of iron per milliliter of culture medium. Because fluctuations in cell populations will also affect the total amount of iron deposited, the data can be normalized and presented as iron/protein ratios. This correction allows the comparison of iron deposition over a parametric iron concentration range. A summary of growth and iron deposition is presented, as mean final iron/protein ratios (after ca. 120 h), with the associated iron concentrations in the left portion of Table 1. The lack of influence of a varying iron concentration upon growth rates and final cell yields has been noted previously (5). The data show a definite increase in the quantity of deposited iron as its concentration is increased in the medium. The ratio increases and reaches a maximum as the iron concentration in the medium is increased from 0.02 to 4.0 mM. It appears that the ability for iron deposition becomes saturated in these laboratory cultures only at an extremely high iron concentration of approximately 2.0 mM. The saturation is alternatively demonstrated by the reciprocals of both the final iron/protein ratios and the associated iron concentrations (right portion of Table 1), which are linearly related (regression coefficient significant at P s 0.001).
Growth in low iron. Because Sphaerotilus seemed to elicit the same growth responses to a wide variety of iron concentrations, no obvious role for the process could be inferred. It seemed possible, however, that this capability TABLE 1. Sphaerotilus, strain SS1, mean final iron/ protein ratios, the associated iron concentrations, and their reciprocals Fe (mM)
0.02 0.10 0.50 1.0 2.0 4.0
Ratio,
0.018 0.17 7.7 15 58 60.5
1/Fe (1/mM)
50 10 2 1 0.5
0.25
1/Ratio°
55.6 5.9 0.13 0.067 0.017 0.0166
Fe/protein (microgram per microgram x 100) after 120 h of incubation. b Protein/Fe (micrograms of Fe/microgram of protein x 100)-'. a
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ROGERS AND ANDERSON
might b4e used to insure adequate amounts of metaboliic iron. To establish the threshold of the iron reqiuirement of strain SS1, cultures were incubate d in medium Ml with no supplemental iron. Thie resulting mediuin, containing approximaltely 0.003 mM iron, did not support growth atfter inoculation and incubation for 136 h. Figurfe 1 is a plot of the growth of two parallel cultuires of equal inocula and incubation conditioras. The 1 mM control curve is derived from a c-ulture containing labeled amino acid and 1 m! M labeled iron from the time of inoculation. Th; at curve representing an iron addition was deri ived from a culture initially containing only L-[3 'H]alanine. The basal iron level of this medium , as with the previous experiment, was approxinnately 0.003 mM. At 54 h after inoculation (arrow), labeled iron was aseptically added to the cl alture, bringing the final iron concentration t;o 1.0 mM, the control level. Growth in the cont rol is similar to previous experiments. The low iron culture displays, at most, a very depresse,d growth until the iron level in the medium is increased. An immediate exponential grovvth period is then observed. Both rates , , , w ' looc)
boc
C
Q) 0 ._c
I
IO FIG.
1
130 90 HOURS .Growth of Sphaerotilus strain SSJ with 50
limiting iron. Control curve represents protein in medium Ml containing labeled 1 mM total iron con-
J. BACTERIOL.
and final yields correspond to those of the control but are delayed by the time preceding the iron addition. Iron deposition in the control and experimental cultures (after iron addition) were both consistent with earlier data. The fact that the organisms fail to grow until after the 0.02 mM iron supplement suggests that the strain's iron requirement is greater than 0.003 mM but not more than 0.02 mM, quite within that range described generally for other gram-negative bacteria (10). It would appear that the capability of iron deposition is of no particular advantage to this organism in sequestering metabolic iron. Trace metals' effect upon final growth and iron deposition. In view of the ability of some Sphaerotilus strains to deposit oxides of manganese as well as iron, the effects of various other trace metals upon the final growth and iron deposition process of strain SS1 were tested. It was thought that this mechanism(s) might provide increased tolerance to toxic concentrations of other trace elements. Five trace metal ions, Co(II), Cu(II), Cr(III), Mn(II), and Ni(II), were chosen because of their proximity to iron in the periodic table (chemical similarity), their essentiality to microorganisms, and their acknowledged toxicity at moderate concentrations (1). Each metal was added to medium Ml containing 1.1 ,ug of iron per ml (0.02 mM) in five 10-fold serial dilutions from 100 to 0.01 ,ug/ ml. The effects of these metals on growth and iron deposition, as percentages of final protein and iron deposited in the control culture (containing only iron), are compared in Table 2 (after 120 h). A general toxic effect at 100 ug/ml can be observed as a marked inhibition of growth in all but the Mn(II) cultures. For the five lower concentrations (0.01 to 10 ,ug/ml), no appreciable inhibition can be demonstrated to result from adding copper, chromium, or manganese. Partial reduction in accumulated cell protein, along with a disproportionately greater decrease in iron deposition, is found with manganese at 100 ,ug/ml, suggesting a competition with iron. However, this competition was very weak, because the manganese was 100 times more concentrated than the iron. Weak competition may also occur with cobalt and nickel at the 10-gg/ml level. No inhibitory couldor be demonstrated effect of any metal to 1 ug/ml. at equal concentrations less than As a basis ffor evaluation, some qualitative . r comparsons of the growth of strains of A. aerogenes, E. coli, and P. denitrificans, in the same medium, supplemented with 1, 10, or 100 ,ug of the trace metals per ml, are provided in Table 3. It is obvious that A. aerogenes and P. denitri-
the outset. The delayed addicell protein (0) of cultures grown in medium Ml, initially containing no labeled supplemE ental iron (basal level of 0.003 mM), and ficans, under these conditions, exhibit a high then labe?led at 54 h (arrow) with 1 mM total iron. tinuousl3 y present from tion curtve represents
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ROLE OF IRON DEPOSITION IN SPHAEROTILUS
267
TABLE 2. Growth and iron deposits of Sphaerotilus strain SSI in medium Ml containing various concentrations of trace metals after 120 h of incubation Ion additiona
Ion concn Protein + % erroe (zg/Ml) (~g/m1)(yg/ml) (~±gIml)
None Co(II)
119 ± 3.8 0.01 112 ± 1 0.10 113 ± 4.4 (1 Lg/ml = 0.017 mM) 1.0 115 ± 3.1 10 77.8 + 3.7 100 NG' CU(II) 0.01 104 ± 2.2 0.10 110 ± 2.0 (1 i.g/ml = 0.016 mM) 1.0 114 ± 4.9 10 129 + 3.4 100 NG Cr(III) 0.01 122 t 2.4 0.10 112 ± 3.9 (1 yg/ml = 0.019 mM) 1.0 112 ± 4.6 10 118 ± 4.6 100 NG Mn(II) 0.01 100 ± 0.9 0.10 108 + 3.2 (1 AgIml = 0.018 mM) 1.0 119 ± 8.5 10 126 ± 1.5 100 86 ± 5.3 Ni(II) 0.01 118 ± 4.0 0.10 109 ± 1.0 (1 jg/ml = 0.017 mM) 1.0 114 ± 3.2 10 79 ± 12.7 100 NG aAll cultures contained 0.02 mM FeC13. b Derived from the standard error of the mean. 'NG, No growth at this metal concentration. ' ND, No iron deposition at this concentration.
tolerance to the metals, growing in every case in concentrations of up to 10 ,ug/ml. All three genera grew at 100 ug of manganese per ml and P. denitrificans exhibited good growth even with 100 ,tg of copper added per ml. E. coli appeared to be somewhat more sensitive, showing inhibition at 10 lug of cobalt and copper per ml. S. discophorus, therefore, fail to demonstrate any exceptional tolerance to the trace elements tested with respect to several other common bacteria. Although the laboratory strains examined may themselves possess above average tolerance to the metals tested, the fact that they do not deposit iron or manganese, as does S.discophorus, does not support the hypothesis that iron deposition in this organism plays an exclusive role in toxic element tolerance. Sphaerotilus could conceivably compete with these genera in a natural mixed microbial population and, as far as the laboratory data indicate, would have no apparent advantage in this respect. Furthermore, the lack of strongly disproportionate effects of all elements, except manganese, upon iron deposition
Percentage of control
protein 100 94.2 95.2 93.3 65.4
87.7 92.7 96.0 109 103 94.5 94.5 99.4 84.4 90.8 99.9 106 72.2 98.7 91.2 95.5 66.9
Fe ± % error
Percentage
of control OAglml) (.gm)Fe
0.146 ± 0.155 ± 0.147 ± 0.167 + 0.066 ± NDd 0.155 ± 0.172 t 0.135 ± 0.167 ± ND 0.174 ± 0.155 ± 0.155 ± 0.155 ± ND 0.159 ± 0.165 + 0.135 ± 0.142 ± 0.0056 ± 0.162 ± 0.176 ± 0.152 + 0.061 ± ND
2.5 7.3 8.9 7.2 3.0
100 106 101 115 45.3
7.8 2.6 16.2 7.0
107 118 92.7 115
3.0 1.8 9.6 2.4
119 107 106 107
5.2 0.3 6.1 4.8 12.4 10 5.2 8.7 83
109 113 93.2 97.6 3.9 112 120 105 41.8
suggests that they do not participate or compete with iron, which has a relatively specific precipitation mechanism. Comparison of iron deposition in selected genera. To establish the relative ability of strain SS1 to precipitate (adsorb) iron, deposition among S. discophorus strain SS1, P. denitrificans, and strain Vl (a S. discophorus strain incapable of manganese oxidation) is compared in Table 4. Iron/protein ratios are used to normalize the differences which might have occurred in final cell mass per milliliter of culture. The ratio for P. denitrificans remains fairly constant during the incubation. Strain Vl exhibits a very low ratio until the final growth point, at which time it attains approximately the same range as that of P. denitrificans. S. discophorus strain SS1 shows its normal high ratio, with final values ranging from 15- to 40-fold greater than those obtained from the strain Vl and P. denitrificans cultures. Because the latter organism produced a pH increase from 7.1 to 7.7 during the incubation period, it is possible that the iron deposition noted in this case can be partly attributed to a
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ROGERS AND ANDERSON
J. BACTERIOL.
TABLE 3. Comparison ofgrowth with added trace metals of different genera Mn (III) Cr (III) Cu (II) Co (II) Strain
1.0" 10 ++b ++
1.0
10
100
1.0
10
100
1.0
10
100
-C ++ Aerobacter aerogenes +d + Escherichia coli ++ Pseudomonas denitrificans ++ ++ a Micrograms per milliliter. b Culture very turbid after 50 h of incubation. c No visible turbidity. d Visible turbidity. eCr(III) caused precipitate at 100 jig/ml.
+ ++
++
++ + ++
++ + ++
*e * *
+ + ++
+ + ++
+ + ++
100
TABLE 4. Comparison of Fe/protein ratios of Sphaerotilus strains SS1 and Vi and P. denitrificans strain DMS-6 Fe/protein ratioa
Strain 39.5 h
48.25 h
64 h
71 h
89.5 h
111.5 hb
5.2 ±8.4 0.036 ± 16.7
5.6 ±4.4 0.065 ± 27.1
7.5 ±4.9 0.42 ± 39.9
17.25 h
41.75 h 48.75 hb
26 h
Sphaerotilus SS1 Vi
0.13
0.43
3.8
+34b
±42.9
±11.4
0.024 ± 71
0.036 ± 38.9
P. denitrificans DMS-6 a Fe/protein ratios given as (micrograms/disintegration per minute) of the mean (percent). b Stationary-phase cells.
purely chemical process. The pH fluctuation has, however, been rigorously examined for strain SS1; the contribution ofthis phenomenon to iron deposition during its growth is negligible. The loss of manganese deposition in strain Vl parallels the loss of iron deposition down to the level of an ordinary (non-iron) pseudomonad. This in turn is strongly suggestive of common causes in the deposition of both ions. Growth at different iron concentrations in dilute media. The use of a "rich" medium for growth of Sphaerotilus may not be an accurate indication of its behavior vis A vis the physiological significance of iron deposition. We questioned, therefore, whether a changing iron concentration could measurably affect final growth of Sphaerotilus in medium containing more dilute organic components, approximating the natural habitat. The effect of successive dilution ofthe organic components (peptone and yeast extract only) of medium Ml upon the final growth yield of Sphaerotilus strain SS1 is shown in Fig. 2. A successive decline in total cell mass with increasing dilution of the carbon and nitrogen source is as expected. To test any effect that a varying iron concentration might have upon final cell yields at the resulting very low growth rates, several diltutions were chosen
x
0.20 0.16 0.13 0.16 ±47.4 ± 4.3 ±12.9 ± 1.8 104 ± variations as standard errors
which produced low, but reliably measurable, growth. The cultures were then incubated in iron concentrations of 0.02, 0.10, and 0.50 mM 120 1-%
80 t I-
.c )
40 H
10
20
30
(Organic Dilution)FIG. 2. Growth of Sphaerotilus strain SS1 in dilute media. Final culture cell protein was estimated from -[3H]alanine incorporation (see Materials and Methods) and plotted versus the reciprocal of the dilution of the organic component (peptone and yeast extract) of medium Ml. Error bars represent the standard error of the mean.
ROLE OF IRON DEPOSITION IN SPHAEROTILUS
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269
TABLE 5. Cellular growth yields of Sphaerotilus strain SSZ as a function of nutrient and Fe(III) dilutions p.g of protein/ml of culture Dilution of orPredicted' ganic componenta
0.02 mM Fe
0.10 mM Fe
0.5 mM Fe
100 103 ± 2.2 94 ± 3.9 20 40 ± 6.8 54 ± 9.4 10 24 ± 14.7 19 2 4 11 ± 14.1 10 ± 10. ld 41.6d 2 4 ± 4.4 6 11.3 57.2 1 0.7 ± 60.7 0.8 ± 39.9 48.5 a (Medium peptone + yeast extract)/(medium Ml peptone + yeast extract). b Assuming that cells yields are simply and directly proportional to concentrations of nutrients regardless of iron concentration. c Second value equals percentage. d Difference statistically significant at the P = 0.05 level. 0 0.20 0.10 0.04 0.02 0.01
93± 44± 17 ± 5 ± 0.5 ± 0.7 ±
5.6"
4.1 3.7
until maximum cell growth was achieved. The resulting cell yields are compared in Table 5. The table lists yields at each of the three different iron concentrations as the organic components of medium Ml are diluted between 0.20 and 0.01 times their normal concentrations. These data represent the results of two different experiments. The individual values comprise a minimum of three and a maximum of six individually inoculated _[3H]alanine-labeled cultures. Pairwise comparisons were made by using the "t" and "F"' tests and the Student-Newmans-Keuls tests on pooled data (0 and 0.04 dilutions). They indicated that, within each dilution, the only (marginally) significant difference (P = 0.05) occurs at the 0.04 dilution, between the 0.02 and 0.10 mM iron cultures. It can therefore be concluded that these data do not support the hypothesis of the proposed differential effect of iron concentration on cell yields for populations growing in rich versus dilute media. DISCUSSION The question posed throughout the investigation concerned the role of the iron deposition process in this organism. The iron/protein ratios, derived from various experiments, indicate the existence of a direct relationship between the extent of iron deposition and the iron concentration in the medium up to a saturation point. The organism's iron deposition capability became saturated only at a high iron concentration of approximately 2 mM. Parenthetically, these saturation data measured a maximum level of iron removal by Sphaerotilus in laboratory culture, which suggests that it cannot exercise significant control over the iron cycle in the natural milieu. Cells growing in a 0.02 mM iron concentration possess this capacity (0.6 mg of iron per mg of cell protein), yet they extracted only about 1.8 x 10-4 mg of iron per mg
of cell protein. The equilibrium, therefore, already favored dissolved iron at this level. A limiting role played by iron-precipitating bacteria in the natural iron cycle (6) is highly unlikely. It was proposed that iron deposition may represent a kind of iron detoxification mechanism for Sphaerotilus. Although far from removing significant quantities of this element, iron deposition could provide protection either by changing the microenvironment around the cell, by rendering the cell and/or its surrounding sheath less permeable to iron, or by preventing access of the toxic metal to the cell membrane. These processes could increase the organism's tolerance to high concentrations of the element. The hypothesis that iron deposition per se is required for a protective function is not supported, because other species of noniron-depositing bacteria (P. denitrificans and E. coli) grew well at 0.5 mM concentrations of this moderately toxic (1) element. The iron requirement of S. discophorus strain SS1 was defined by experiments measuring growth at low iron concentrations. We established the minimal requirement as being in the 0.003 to 0.02 mM range. This is similar to the requirement of E. coli growing on acetate (Neidhardt, personal communication) and suggests that the iron deposition mechanism is not particularly advantageous to this organism in satisfying the requirement. In the comparisons of iron deposition capability, Sphaerotilus strain SS1 demonstrates quantitatively the well-known distinction of the iron-depositing bacteria from other bacteria by its ability, in the laboratory, to attain iron/ protein ratios between 10- and 100-fold greater than those observed in S. discophorus strain Vl and in P. denitrificans cultures. It is probable that S. discophorus strain Vl (nonmanganese-depositing strain), as evidenced by its
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ROGERS AND ANDERSON
observed morphological and physiological characteristics, is identical to the S strains of S. discophorus, which were isolated by Stokes and Powers (8). These strains were identical to the wild-type strains, with the exception that the S strains lacked the ability for sheath production and for manganous salt oxidation. Microscopy examinations suggested that sheath production by S. discophorus strain Vi, compared to strain SS1, is absent, or at least minimal. Strain Vi demonstrated a reduced capacity to deposit iron, which was commensurate with that of P. denitrificans, a non-iron bacterium, although it could still grow normally in relatively high iron (1.5 mM). This evidence supports the proposed link between sheath production and manganese and iron deposition and, furthermore, does not confirm the hypothesis of iron tolerance conferred by the process. A criticism could be leveled at the extrapolation of the findings of a laboratory culturing system to the organism's behavior in the environment. Results obtained with dilute media demonstrate that the use of the in vitro system probably does not create misleading information concerning iron concentration and its inability to affect the yield of S. discophorus cells. When the organism's growth rate was significantly decreased by the nutrient limitation imposed by diluting the peptone and yeast extract components of the media, no significant differences in the final cell yields could be demonstrated at iron concentrations between 0.02 and 0.50 mM. Metal deposition in Sphaerotilus was hypothesized as being a detoxification mechanism, which might provide the bacterium with a resistance to growth inhibition caused by high concentrations of certain trace elements. We questioned whether other elements, with chemical properties similar to those of iron, could interfere with the process used for iron deposition, and whether S. discophorus strain SS1 was unusually resistant to the toxic effects of these ions. Salts of cobalt, copper, chromium, manganese, and nickel were added to the cul.. ture medium so that their effect upon iron deposition and growth could be observed. Athough SS1 did show a tolerance to these metals at concentrations up to 10 ,ug/ml, the comparison to other genera indicated nothing exceptional about the response. The results indicated that strain SS1 is of equal or greater susceptibility to growth inhibition by these metals, compared to other selected microbes. Sphaerotilus growth and iron deposition was also observed in the presence of manganous ions. The strain comparison indicated that a 100-,ug concentration of
J. BACTERIOL.
manganese per ml is not inhibitory to the other microbes which were compared to Sphaerotilus. The disproportionate decrease in iron deposition of Sphaerotilus (to 3.9% of the control), accompanied by growth that was depressed to 72.2% of the control, indicated preferential inhibition of iron uptake, although selectivity for iron is apparent by the magnitude of Mn(II) necessary to achieve this inhibition. The competitive effect was achieved only after manganese was supplied in quantities which were almost 100-fold greater than those of iron (0.02 mM iron and 1.82 mM manganese). This result is not consistent with a deposition mechanism for which the principle substrate is manganese; therefore, iron precipitation is not a simple byproduct of manganese oxidation. Oxidation of manganese, however, might be favored in Sphaerotilus growing under certain conditions of low oxygen tension in the presence of sulfide. It is possible that the same phenomenon may occur when either Co(II) or Ni(II) is added (10 ,ug/ml or approximately 0.2 mM), although the disproportional decreases of iron deposition and growths were less pronounced. Reciprocal experiments (iron inhibition of labeled manganese oxidation) are necessary to establish the duality of the mechanism. S strains of S. discophorus, which arose by an unknown consequence of heat shock (see Materials and Methods), apparently have lost both iron and manganese deposition capabilities. This fact suggests relationships between the two, but until the basis for the conversion is known it would be premature to ascribe both losses to a simple genetic (mutation?) event. The fact that S strains have identical growth rates and yields as their parent again illustrates the lack of a gross physiological role for ion deposition. Mulder (2) and van Veen (9) proposed that manganese oxidation was protein facilitated, and that this process exhibited a behavior which was characteristic of an enzyme-catalyzed reaction. The present investigation suggests that iron deposition and manganese oxidation might share the same mechanism. We speculate that a moiety of the organism's sheath is responsible for catalyzing the deposition of both manganese and iron, although the specific function of this phenomenon is elusive. ACKNOWLEDGMENTS Financial support for this work was provided in part by the Cramer Foundation of Dartmouth College and by Sigma Xi Grants-in-Aid for Research. LITERATURE CITED 1. Bowen, H. J. M. 1966. Trace elements in biochemistry. Academic Press Inc., New York.
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ROLE OF IRON DEPOSITION IN SPHAEROTILUS
2. Mulder, E. G. 1972. Le cycle biologique et aquatique du der et du manganese. Rev. Ecol. Biol. Sol. 9:321-348. 3. Mulder, E. G., and W. L. van Veen. 1963. Investigation
the Sphaerotilus-Leptothrix group. Antonie van Leeuwenhoek J. Microbiol. Serol. 29:121-153. 4. Pringsheim, E. G. 1949. The filamentous bacteria Sphaerotilus, Leptothrix, and Cladothrix, and their relation to iron and manganese. Phil. Trans. R. Soc. London Ser. B 233:453-482. 5. Rogers, S. R., and J. J. Anderson. 1976. Measurement of growth and iron deposition in Sphaerotilus discophorus. J. Bacteriol. 126:257-263. 6. Ruttner, F. 1952. Iron and manganese, p. 83-87. In D. on
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G. Frey and F. E. J. Fry (ed.), Fundamentals of limnology. University of Toronto Press, Toronto. 7. Starkey, R. L. 1945. Transformations of iron by bacteria in water. J. Am. Water Works Assoc. 37:963-984. 8. Stokes, J. L., and M. T. Powers. 1965. Formation of rough and smooth strains of Sphaerotilus discophorus. Antonie van Leeuwenhoek J. Microbiol. Serol. 31:157-164. van Veen, W. manganese
L. 1972. Factors affecting the oxidation of by Sphaerotilus discophorus. Antonie van Leeuwenhoek J. Microbiol. Serol. 38:623-626. 10. Weinberg, E. D. 1974. Iron and susceptibility to infectious disease. Science 194:952-956. 9.
