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Keywords:

  • Colorless sulfur bacterium;
  • Lithoheterotrophy;
  • Cytochrome;
  • Sulfur metabolism;
  • Metabolic regulation;
  • Leucothrix mucor

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

Evidence is presented that the type strain of filamentous gliding bacterium Leucothrix mucor DSM 2157 is capable of lithoheterotrophic growth. Thiosulfate oxidation was accompanied by accumulation of sulfate and tetrathionate in the growth medium and intracellular accumulation of elemental sulfur, as occurs in filamentous sulfur bacteria of the genus Thiothrix. Thiosulfate oxidation by L. mucor was induced during growth with a limiting concentration of organic substrate (i.e. 50–170 mg l−1 tryptone) and resulted from the induction of dissimilatory enzymes of sulfur metabolism. Metabolically useful energy was derived by both substrate-linked and oxidative phosphorylation from thiosulfate and sulfite oxidation. During sulfite oxidation the electron transfer pathway was shown to begin at the ubiquinone-cytochrome b segment of the electron transport chain. L. mucor limited by organic substrate had lowered levels of membrane bound cytochromes c and respiratory activity but a higher input of a cyanide-insensitive respiratory component than during organic enriched growth. Cytochromes aa3 and d, which had not been shown previously in this bacterium, were detected in the cells under various growth conditions. Cytochrome aa3 is suggested to take part in the electron transfer pathway from thiosulfate to oxygen which was completely inhibited by 100 μM cyanide.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

Leucothrix mucor is a representative of filamentous gliding bacteria, a typical epiphyte living in the marine tidal zone on higher marine plants, macroalgae, benthic invertebrates and fish eggs [1–3]. Massive fouling of gills by L. mucor might be the reason for crustacea death in marine culture [4]. Development of this strain in the consortium of filamentous sulfur bacteria is often associated with bulking in the cleaning constructions for sewage [5].

Early investigations of H2S oxidation by several L. mucor strains gave negative results [6]. Later, K. Eimhjillen found sulfide and thiosulfate oxidation by the pure L. mucor culture isolated at Pacific Grove, California [7]. However, until now no evidence for any energy link has been found and Brock [7] pointed out that more detailed investigations on using sulfur compounds in L. mucor metabolism are required.

Until recently L. mucor was the only species of the genus. In 1996 the second representative, Leucothrix thiophila, was described [8]. It uses sulfur compounds in energy metabolism and in this respect, and in its similar development cycle, it resembles the filamentous sulfur bacteria of the Thiothrix genus. The aim of the present study was to detect whether the L. mucor type strain can oxidize sulfur compounds and to define biochemical pathways involved in lithoheterotrophic growth.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

2.1Bacteria and growth conditions

The type strain of L. mucor DSM 2157 (ATCC 25107) was received from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (GmbH, Braunschweig, Germany). The organism was grown in the following modified medium [9] containing (g l−1): NaCl 24, Na2SO4 4, KCl 0.7, BaCl2 0.1, Na2SeO3 0.005, NaF 0.003, SrCl2 0.04, NH4NO3 0.002, FeCl3 0.001, HBrO3 0.03, distilled water, pH 7.8. After autoclaving the basal medium at 121°C for 15 min, sterile solutions of MgCl2·7H2O and CaCl2·2H2O were added to final concentrations of 11 g l−1 and 2 g l−1, respectively. When required a sterile solution of tryptone (Ferak, Germany) was added to a final concentration of 0.05–2 g l−1. Sodium thiosulfate pentahydrate was sterilized by ultrafiltration and added to a final concentration of 1–3.3 g l−1 as indicated in the text.

2.2Assays of enzyme activity

Cell extracts were prepared from the bacteria using a UZDN-2T sonicator at 500 W and 22 kHz for 3 min in an ice-cold bath. Activities of sulfur metabolism enzymes in the cell extract supernatant were measured spectrophotometrically as described earlier [10]. L. mucor membranes were obtained using a method described earlier [11] by disruption of the cells in a French press. Respiratory activity of the cell suspension and membranes was measured using a standard Clark-type oxygen electrode at 25°C.

2.3Analytical procedures

H2S was determined by a colorimetric method using dimethyl-n-phenylenediamine [12], or by iodometric titration. When the products of thiosulfate metabolism were not measured, thiosulfate was analyzed by the iodometric method upon sulfite binding to formaldehyde [13]. On measuring the products of thiosulfate metabolism, thiosulfate, tetrathionate and trithionate were determined separately by the cyanolytic method [14], elemental sulfur by the method of Morris et al. [15], and sulfate by the chloranilate method as described previously [16]. Protein was determined by the method of Lowry [17]; in the case of bacterial cell protein the procedure was used following the removal of elemental sulfur from centrifuged cells by ethanol extraction for an hour followed by buffer washing. The amino acid content of the growth medium after removal of the cells from the medium by ultrafiltration was measured both by the method of Lowry [17] and with ninhydrin [18] using BSA as a standard. SDS-PAGE was performed according to the Laemmli method [19]; the gels were stained as described by Thomas et al. [20] to detect covalently bound heme. The absorbance difference spectra of membranes were recorded with an SLM Aminco DW-2000 spectrophotometer at room temperature [11]. Unless otherwise indicated, values in the tables and figures represent the mean of three determinations. Standard deviations were less than 5% of the mean. Each experiment was repeated at least 2–5 times and representative experiments are shown in the figures.

3Results and discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

3.1Thiosulfate oxidation and its effect on bacterial growth

On cultivation of L. mucor in the medium containing 270 mg l−1 tryptone (Figs. 1, 2), oxidation of thiosulfate began only after a lag, presumable due to an initial depletion of the basic pool of assimilated organic compounds. Before thiosulfate oxidation began, the concentration of amino acids in the growth medium fell to 100–170 mg l−1 (Fig. 1A). The Lowry method might overstate tryptone content in the growing culture compared to the ninhydrin method (Fig. 1A) presumably because of the less rapid metabolism of aromatic amino acids or the accumulation of products of cell metabolism (extracellular polysugars) during bacterial growth. In the medium containing 50 to 100 mg l−1 tryptone and 3 g l−1 thiosulfate, bacterial growth was immediately accompanied by thiosulfate oxidation and declined on depletion of organic substrates (Fig. 1B).

image

Figure 1. A: Effect of thiosulfate on tryptone-dependent growth of L. mucor DSM 2157 culture: ◯, thiosulfate oxidation; □, tryptone consumption measured by the Lowry method and ?, with ninhydrin; ▾, the cell protein increase in the presence of thiosulfate in the growth medium; ▿, the same as in ▾ but in the absence of thiosulfate. In a separate experiment the cell protein increase was measured in the absence of thiosulfate in the same growth medium. B: Thiosulfate oxidation in the medium containing initially 50 mg l−1 tryptone. For symbol legends see A. The results of representative experiments are presented in A and B.

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image

Figure 2. Accumulation of the products of thiosulfate oxidation in the culture of L. mucor DSM 2157. A: ♦, S/S2O32− mg l−1; ▴, S/So42− mg l−1. B: ◯, S/S4O62− mg l−1; □, S0 mg l−1. In experiments when the thiosulfate oxidation products were measured the initial concentration of Na2SO4 was 0.7 g l−1 instead of 4 g l−1.

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Thiosulfate oxidation was accompanied predominantly by accumulation of sulfate in the medium and to a lesser extent tetrathionate (Fig. 2); the latter was oxidized to sulfate in the stationary phase of growth. Accumulation of elemental sulfur only occurred intracellularly, similar to that in filamentous sulfur bacteria of Beggiatoa and Thiothrix genera. Note that it is difficult to differentiate morphologically Leucothrix and Thiothrix rosettes and filaments containing intracellular inclusions of sulfur. Balance calculations of thiosulfate oxidation products showed sulfur disappeared in the cells at the end of the stationary phase of growth and, like tetrathionate, was oxidized to sulfate. Thiosulfate oxidation was always accompanied by a slight increase in bacterial yield compared to the case of cultivating the bacteria in the absence of thiosulfate (Fig. 1A).

It is not known why the yield of bacteria in the presence of thiosulfate was higher than in its absence even before a decrease in the thiosulfate concentration in the culture could be detected (Fig. 1A, first 48 h of the experiment). However, in 15 experiments, the rate of tryptone consumption per unit of biomass in the presence of thiosulfate was consistently about half that either in its absence, or during the initial phase of growth in the presence of thiosulfate. This strongly suggests that energy is conserved, and hence that L. mucor can grow lithoheterotrophically, in the presence of thiosulfate.

3.2Sulfur metabolism enzymes

During growth with a low tryptone concentration (0.2 g l−1) and thiosulfate in the cultivation medium, the biosynthesis of the sulfur cycle enzymes was induced by one to two orders. Attention is drawn to the sharp increase in activity of sulfite oxidase (sulfite ferricyanide oxidoreductase), thiosulfate oxidase (thiosulfate ferricyanide oxidoreductase) and, especially, APS reductase which are the dissimilatory key enzymes of sulfur compound oxidation (Table 1). The activities of the mentioned enzymes are close to those known for representatives of a number of groups of chemolithotrophic bacteria [21,22].

Table 1.  Activity of enzymes metabolizing sulfur compounds in L. mucor DSM 2157
  1. aEnzyme activities are the average of three to five independent experiments. Standard deviations were usually less than 5%. Values represent the mean of triplicate determinations and standard deviations were usually less than 5%. In the case of a wide variation in the magnitude of activities the limiting values are specified.

Enzymesnmol min−1 (mg protein)−1a
 Growth conditions
 2 g l−1 tryptone0.2 g l−1 tryptone
 +S2O32−−S2O32−+S2O32−
Sulfite ferricyanide oxidoreductase015380
Thiosulfate ferricyanide oxidoreductase0590
APS-reductase08160–580
Rhodanese2660–10886–145

3.3Interrelation between enzymatic oxidation of sulfur compounds, electron transport chain, oxidative and substrate-linked phosphorylation

Sulfite oxidation was characterized by a high respiratory activity (Table 2), which varied from 46 to 253 nmol O2 min−1 (mg protein)−1 and decreased intensively in the freshly prepared cell suspension. The tested inhibitors, except rotenone and N-ethylmaleimide (NEM), completely arrested sulfite-dependent bacterial respiration. The inhibitory effect of myxothiazol and antimycin A on the cell respiration shows that electrons from sulfite oxidation enter the electron transport chain at the ubiquinone-cytochrome b segment. Assuming participation of an a-type oxidase (see Section 3.4) in sulfite oxidation which was inhibited by 100 μM cyanide (Table 2), the process should be characterized by two coupling points of energy conservation. High activity of APS reductase on thiosulfate oxidation (Table 1) indicates that energy generation in L. mucor cells should occur by substrate-linked phosphorylation as well as by oxidative phosphorylation.

Table 2.  Effect of inhibitors on sulfite-dependenta respiration of L. mucor DSM 2157 cells
  1. aSulfite was added at 20 mM.

  2. bIn this case K1 and K2 were subtracted from O2 consumption rate where K1 (endogenous respiration) was close to zero and K2 (sulfite autooxidation) varied in the range of 0–1.9 nmol min−1 (mg protein)−1.

InhibitorInhibitor concentration, μMO2 consumption rate, nmol min−1 (mg protein)−1Inhibition, %
  −inhibitor+inhibitorb 
Rotenone50.094.094.00
Antimycin A90.046.30.0100
Myxothiazol25.0253.00.0100
KCN100.092.70.0100
NEM50.092.746.350
The cells were grown to the exponential phase of growth in the medium containing 0.2 g l−1 tryptone and 2 g l−1 thiosulfate.
Values represent the mean of triplicate determinations; standard deviations were less than 0.5%.

3.4Cytochrome composition

Cytochromes b, c and o but no cytochromes a were previously found in L. mucor during organoheterotrophic growth [23]. We showed that cytochrome a (α-band at 605 nm; Fig. 3, dotted line) was present in L. mucor membranes in the same concentration under various growth conditions (Table 3). Evidently, it is an aa3-type oxidase as one of the hemes a bound cyanide (Fig. 3, solid line). Cytochromes b composition and function in the electron transport chain are of special interest as their content decreased by 30% when the bacteria were grown with the low tryptone concentration (0.2 g l−1) in the absence of thiosulfate (Table 3). Horse heart cytochrome c as well as sodium ascorbate in the presence of TMPD were oxidized by the membranes, the respiratory activity with the latter substrate being about 8 μmol O2 min−1 (mg protein)−1 during tryptone-enriched growth and of about one order lower magnitude under tryptone-limited growth. This fact is in good correlation with the much higher content and diversity of membrane cytochromes c in tryptone-enriched growth conditions (Table 3). Cyanide inhibited respiratory activity by 70 and 60% in tryptone-enriched and tryptone-limited conditions, respectively, with apparent Ki of 5 and 15 μM. The remaining respiratory activity appeared to be insensitive to cyanide at a concentration of about 0.5 mM. The presence of this 0.5 mM cyanide-insensitive component in membrane respiration might be due to participation of a cytochrome d (α-band at 626 nm; Fig. 3, dashed line and inset) which has never been identified in this bacterium. This suggestion does not exclude the presence of an additional, authentic, cyanide-resistant oxidase like the one shown in Pseudomonas aeruginosa[24].

image

Figure 3. Reduced-minus-oxidized difference spectra of L. mucor DSM 2157 membranes isolated from the cells harvested after lithoheterotrophic growth. One mM cyanide reduced-minus-air oxidized difference spectrum (solid line); dithionite reduced-minus-air oxidized difference spectrum (dotted line); dithionite reduced-minus-1 mM cyanide reduced difference spectrum (dashed line). The upper inset at the right of the illustration demonstrates cytochrome d presence at the difference spectrum of dithionite reduced L. mucor membranes minus dithionite reduced purified bovine heart aa3-type oxidase which was added in the separate cuvette in equimolar ratio to L. mucor aa3-type oxidase.

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Table 3.  Cytochrome content in L. mucor membranes in various growth conditions
  1. acm, membrane bound cytochrome c.

  2. bcs, soluble cytochrome c.

Growth conditionsCytochrome content in nmol heme (mg protein)−1
 a605b557dcma  csb
    37 kDa25 kDa20 kDa 
0.2 g l−1 tryptone+S2O32−0.088±0.0080.60±0.01+n.d.n.d.n.d.+
0.2 g l−1 tryptone−S2O32−0.095±0.010.42±0.02++++n.d.n.d.+
2 g l−1 tryptone0.078±0.0080.63±0.02+++++++
To calculate cytochrome content the following millimolar extinction coefficients were used: 13 at 605–630 nm for cytochrome a and 17.5 at 557–575 nm for cytochrome b. Membrane bound cytochromes c were detected by staining the gels after SDS-PAGE as described in Section 2. This procedure allows to identify cytochromes c only because heme c in contrast to other hemes is bound covalently to proteins and retained in gels in SDS presence.
n.d., not detectable.

Based on the data on the dynamic of thiosulfate oxidation products and sulfur metabolism enzyme activity the following biochemical mechanism of oxidation by L. mucor DSM 2157 of thiosulfate and other sulfur containing compounds might be suggested: inline image 1, rhodanese; 2, thiosulfate splitting complex; 3, sulfide oxidase (not determined); 4, sulfur oxygenase; 5, sulfite oxidase; 6, APS-reductase; 7, ADP-sulfurylase (not determined); 8, thiosulfate oxidase; 9, sulfur reductase. Wide arrows show the high activity of the corresponding enzymes. ADP-phosphorylase (7) was not determined but as a rule it takes part in ATP production simultaneously with APS-reductase. The dashed line indicates a reaction for which the enzyme was not studied. However, the declining tetrathionate content in the medium was shown to be accompanied by the enhanced sulfate content (Fig. 2).

Comparative analysis of sulfur metabolism of L. mucor DSM 2157 and of filamentous colorless sulfur bacteria of Beggiatoa and Thiothrix genera shows some essential differences in functional significance, biochemical mechanisms of sulfur compound oxidation and energy coupling. The energy obtained by B. leptomitiformis D402 on thiosulfate oxidation is strictly coupled to oxidative phosphorylation [10], while the representatives of Thiothrix genus –Thiothrix ramosa and Thiothrix arctophila IN – use substrate-linked phosphorylation only [22,25]. In contrast to the above mentioned sulfur bacteria, both substrate-linked and oxidative phosphorylation function in L. mucor DSM 2157 as well as in L. thiophila[25]. Capability for lithoheterotrophy of L. mucor strain earlier believed to be an organotrophic organism is a useful character in some natural habitats where the availability of accessible organic substances might be limited. It is necessary to emphasize that the limitation of the bacterium by the organic growth substrate induces activity of sulfur metabolism enzymes and in this way makes it possible to obtain an additional source of energy and gives an advantage in competition with other microorganisms in the natural community. The finding of lithoheterotrophy of L. mucor allows extension of microorganisms participating in the transformation of sulfur compounds in natural environments.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

The authors express gratitude to Professor J. Cole for critically reviewing the manuscript and helpful advice. The research was supported by grants from the Russian Foundation for Basic Research (grant 99-04-49161 and 96-04-50-938), the State Scientific and Technical Program ‘Biodiversity’ 2.1.2 and in part by the State Technological Program of Russia ‘New methods in Bioengineering’ (grant 03.0001H-316).

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References
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