The role of benzoate secreted by Desulfotomaculum acetoxidans DSM 771 in sulfate uptake

This work was designed to find the cause of the delay in hydrogen sulfide dissimilation in Desulfotomaculum acetoxidans DSM 771, which is dependent on the sulfate uptake. This bacterium grown without addition of any aromatic compound was shown by spectrum analysis with the methylene method to contain hydroxy-benzoate derivatives. The presence of these compounds was confirmed by HPLC in fractions obtained from cell walls after 15 days of culture. The test with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt seemed to indicate the presence of peroxidase, which probably oxidized benzoate to its hydroxy derivatives. The test with 5-sulfo-salicylic acid proved the ability of the investigated strain to utilize arylsulfates and to reduce sulfate group to hydrogen sulfide. On the basis of the above data, we propose the following sequence of reactions: 1, benzoate secretion; 2, benzoate hydroxylation; 3, sulfonation of hydroxy-benzoate derivatives.


Vol. 52 No. 4/2005, 797-802
on-line at: www.actabp.pl The sulfate-reducing bacteria (SRB -group 7) (Widdel & Pfennig, 1984) are capable not only of sulfur assimilation but also of dissimilative reduction of sulfate or sulfur.This specific pathway is coupled with utilization of hydrocarbon derivatives (e.g.lactate): However, the species Desulfotomaculum acetoxidans is also capable of complete oxidation of acetate, which can contribute to survival of these bacteria.
Sulfate transport has been the most comprehensively investigated in Gram-negative bacteria (e.g., Escherichia coli and Salmonella sp.) (Silver & Walderhaung, 1992).In these bacteria, sulfate transport into the cell is catalyzed either by ATP-binding casse�e (ABC)-type transporters (SulT family) or by major facilitator superfamily-type transporters (SulP family) (Kertesz, 2001).Unfortunately, sulfate uptake processes in SRB are still very poorly understood although these bacteria obtain energy also from the anaerobic respiratory chain of sulfate metabolism.On the basis of the increase in hydrogen ion concentration accompanied by a simultane-ous decrease only in sulfate level, Cypionka (1987) concluded that sulfate uptake by the sulfate-reducing bacteria took place by a symport with hydrogen protons.Several researchers confirmed the lowering of the media pH during the first days (1-3) of culture in the lag phase (Cypionka, 1989;Pado et al., 1994). However, Raskin et al. (1996) investigating co-cultures of sulfate-reducing and methanogenic bacteria found that such microorganisms could survive in culture for over two years.Interestingly, the addition of sulfate a�er such a long period did not cause immediate production of H 2 S.An increased H 2 S level was only found in log and stationary phases (not earlier than a�er 3-4 days of culture), most probably as a product of a dissimilatory pathway (Pado & Pawłowska-Ćwięk, 2004).The question arose of what caused the delay in H 2 S production when the sulfate reduction process (especially at the bisulfite step) proceeded at a high rate (Postgate, 1984;LeGall & Fauque, 1988;Moura et al., 1988).
The above findings prompted us to make an a�empt to explain the reasons of the delay of H 2 S dissimilation.Desulfotomaculum acetoxidans (subgroup 1) was selected for the tests, because it is a Gram-positive bacterium and thus is a more convenient object of laboratory studies than Gram-negative SRB (e.g., Desulfovibrio sp.), which require of additional purification procedures.

Growth of organisms.
Cultures of Desulfotomaculum acetoxidans DSM 771 were maintained on a medium containing (per 1 dm 3 ): 1.15 mM KH 2 PO 4 , 4.02 mM KCl, 5.61 mM NH 4 Cl, 1.13 mM CaCl 2 , 1.97 mM MgCl 2 , 85.55 mM NaCl, trace elements (according to DSM-bank prescription) and additionally 21.12 mM sodium sulfate and 42.24 mM sodium lactate as substrates; without any addition of aromatic compounds; pH of the media were adjusted to 7.0 ± 0.2.The autoclaved and inoculated media were immediately covered with a liquid paraffin layer 3-5 mm thick.Some cultures were grown with the addition of 21.12 mM 5-sulfo-salicylate without lactate and sulfate.All cultures were grown at room temperature (19-24 o C).
Turbidity of cultures was measured at 436 nm (Cypionka, 1987) and the growth rate was estimated from turbidity measured on consecutive days of culture (τ t n ) relative to the initial turbidity of that culture (τ t 0 ).
Cell wall preparation procedure.On the 1st and 15th culture day, 1 dm 3 aliquots were withdrawn and centrifuged at 2500 × g for 25 min.Cell biomass pallets were thrown away, while the supernatants were evaporated with several crystals of sodium dithionite (Na 2 S 2 O 4 ) to about 50 ml at 8 o C. The separated cell wall pellets were centrifuged at 10 000 × g for 15 min and lysozyme solution (10 5 U/1 cm 3 10 mM Tris buffer, pH 6.9) was added to the pellet to degrade peptidoglycan.The suspension was shaken and incubated for 48 h and recentrifuged.Then the pellets were extracted with 2 mM piperazine-N,N'-bis(2-ethane-sulfonic acid) (Pipes) buffer (pH 5.5) for 24 h.This operation was repeated three times and the extracts were pooled.
The supernatants following the Na 2 S 2 O 4 treatment were evaporated to form crystals and were treated with 100 cm 3 of methanol for 8 h and centrifuged as previously.
Spectrophotometric analysis.The colorimetric and spectral analyses were performed using a CECIL 8020 spectrophotometer (bandwidth 2 nm and path length 10 mm), and data were processed with the computer program DataStream-Plus (both Cecil Instruments Ltd).
The chemicals and reagents (except for 4-hydroxy-3-sulfo-benzoate) were obtained from Merck, Fluka or Sigma Chemical Companies.

RESULTS AND DISCUSSION
Growth rate and H 2 S (and/or sulfide) level during incubation of Desulfotomaculum acetoxidans are presented in Fig. 2.
Several researchers have proven that some species of sulfate-reducing bacteria are able to degrade derivatives of amine-, methyl-, and hydroxy- The role of benzoate secreted by Desulfotomaculum acetoxidans benzene (Schnel et al., 1989;Schink et al., 1992;Gorny & Schink, 1994;Rabus & Widdel, 1995).Kuever et al. (1993) claimed that a strain called Groll (a new species of the genus Desulfotomaculum) utilized benzoate and 4-hydroxy-benzoate so fast that the lag growth phase was not observed at all, whereas the degradation of 3-hydroxy-benzoate and 3,4-dihydroxy-benzoate needed more than 60 h.Utilization of other aryls, among them phenol, o-dihydroxy-benzene, mand p-cresol and hydroxy-benzylalcohols required even up to 8 days.Since benzoate and 4-hydroxybenzoate are utilized with similar easiness and velocity, two basic questions need to be answered: which enzyme catalyzes the hydroxylation of benzoate and whether this reaction proceeds inside or outside the cytosol.
Samples from the first days of incubation (lag phase) temporarily turned pink (salmon) when reagents A and B, used for the determination of H 2 S, were added (see chemical tests).This prompted us to perform a spectral analysis.Spectra were obtained in the range 400-700 nm for samples collected a�er 24, 48 and 72 h of culture and treated with reagents A and B. They revealed two clear maxima at about 410 nm and about 665 nm and a much less pronounced one at about 505 nm (Fig. 3).It was very interesting that during the study period the peak at about 410 nm decreased whereas the peak at about 665 nm increased.The maximum at about 665 nm is characteristic of methylene blue, which is a product of the reaction of hydrogen sulfide with the reagents used in the method.Obviously, during the consecutive days of incubation the absorbance at 665 nm was permanently increasing, which proved an increased level of H 2 S.These results correspond well with our earlier research concerning the beginning of the dissimilatory pathway (Pado & Pawłowska-Ćwięk, 2004).The delay of H 2 S dissimilation can be a result of the metabolic priority of an assimilation pathway of sulfate reduction, however, other causes should not be disregarded.
Most of the standards (4-hydroxy-benzoate, 5sulfo-salicylate and 4-hydroxy-3-sulfobenzoate -see Materials and Methods), but not benzoate, formed complexes with Fe 3+ present in reagent B with the maxima in their absorption spectra at 411.7 nm, 503.5 and 501.2 nm, respectively (Fig. 4).Based on these data, the results of bacterial cell wall analysis most likely suggest the presence of 4-hydroxy-benzoate whose amount seems to decrease progressively a�er 24 h of incubation (Fig. 3).The small peak at about 505 nm can indicate the presence of a sulfate derivative of 4-hydroxy-benzoate rather than salicylate (compare Figs. 3 and 4).
The methanol extract from crystals obtained from the 15-day culture was fractionated by HPLC.The obtained fractions were subjected to spectral analysis and chemical tests.Peaks of initial fractions (9-16) had absorbance maxima in the range 253-262  1 (1, with maxima at: 407.2, 410.9 and 512.0 nm), 2 (2, 411.2; 520.1 and 667.0 nm), and 3 (3, 503.2 and 666.7 nm) days samples with methylene blue method's reagents N,N-dimethyl-p-phenylenediamine (reagent A) and Fe(NH 4 )(SO 4 ) 2 (reagent B).The obtained results indicate the presence of at least two compounds of that type (Fig. 5).The phenolic character of those compounds was confirmed by preliminary identification with chromic acid.Fractions obtained by repeated HPLC were treated with reagent B. The absorption spectra showed the presence of ferrous complexes with 4-hydroxy-3-sulfobenzoate (fraction 10) and 4-hydroxy-benzoate (fraction 11) (Fig. 6).The poorly distinguished peak at 502 nm (fraction 10) may suggest fast transport of 4-hydroxy-3-sulfo-benzoate into the cytosol.
The UV/VIS spectrum of the Pipes buffer extract from the cell wall precipitate from the 1-day culture was characterized by maxima at 417, 525 and 551 nm (Fig. 7).The test with the use of ABTS and H 2 O 2 , which are specific reagents for peroxidase, was performed (Fig. 8).The obtained results seem to indicate that the protein exhibiting peroxidase activity which appeared in the investigated bacteria is similar to horseradish peroxidase.This cell wall protein, and particularly its catalytic specificity, requires further research.The hydroxyl group position seems to be important since 4-hydroxy-benzoate was utilized faster than 3-hydroxy-benzoate (Kuever et al., 1993).One should remember that 4-hydroxybenzoate is a product of an enzymatic reaction, while 3-hydroxy-benzoate may be produced in other ways as well.Probably the presence of a carboxylic group is also very important, because the growth of this bacterium on media with an aryl compound but without a -COOH group (e.g., phenol) is poor (Kuever et al., 1993).The carboxylic group binding to transport proteins could make transport processes into the cytosol easier and faster.
There must be a reason for the secretion of a high-energy metabolite, such as benzoate, into the    The role of benzoate secreted by Desulfotomaculum acetoxidans cell wall by the investigated strain.The possibility of sulfonation of hydroxyaryls is supported by the following facts: 1. Sulfonation of hydroxy-benzoates runs easily in the laboratory; 2. Lowering of pH facilitates sulfonation and such lowering is characteristic of the lag phase; 3. The peak appearing at �505 nm proves the presence of a 4-hydroxy-3-sulfo-benzoate complex with iron (compare Figs. 3 and 4).
Considering the above observations, 2-hydroxy-5-sulfo-benzoate (5-sulfo-salicylate) was used as the only source of sulfates and carbon in the growth medium.However, the growth rate and H 2 S level increased earlier and quicker in a culture with lactate plus sulfate than in one with 5-sulfo-salicylate (Fig. 2).These results indicate that uptake (and maybe assimilation too) of 5-sulfosalicylate requires the longest time.The H 2 S level (Fig. 2) and absorption spectra (Fig. 9) showed a steady increase starting from the 13th day of incubation.This leads us to believe that the investigated species has the ability of utilizing the sulfate group as a substrate for the sulfate reduction pathway.This supports the possibility of sulfonation reaction of hydroxy-benzoates.A significant prolongation of the delay of H 2 S dissimilation when the culture was carried out with 5-sulfo-salicylate, in comparison with any monoaryl derivative, might indicate the necessity of shi�ing of the hydroxyl group from position 2 to position 4 in the ring.
The obtained data suggest the following sequence of processes occurring in cell walls during the lag phase of growth: 1. Secretion of benzoic acid or its derivatives (this step is not needed if those compounds are directly added to the medium); 2. Oxidation of the ring, probably in position 4 (if 4-hydroxy-benzoate is added to the medium, this step is also not needed); 3. Sulfonation of 4-hydroxy-benzoate in the free 3-or 5-positions in the ring (Fig. 10).
When the first step or both the first and the second one are not necessary, the lag phase might be shortened so considerably that it would become unnoticeable.It can be reasonably assumed that the kinetics of the third step is much faster than that of the first two steps.However, when sodium sulfite instead of sodium sulfate was added to the nutrient medium, the lag phase became longer by oneweek (unpublished).This could be considered as an additional argument supporting the sulfonation process, for which sulfites (HSO 3 -) are not appropriate substrates.The sulfate uptake by sulfonation of 4-hydroxy-benzoate could be specific but more efficient than other ways for sulfate transport.Pseudomonas putida S-313 strain, which is able to consume monoaryls, was found to contain a specific transporter protein (AsfC or AtsB) binding arylsulfonates (Vermeĳ et al., 1999).Other microorganisms obtain sulfur either from inorganic sulfate or from organosulfur compounds such as arylsulfonate (Valdés et al., 2003).
In the cytosol, 4-hydroxy-3-sulfo-benzoate is most probably hydrolytically desulfonated and the sulfate group becomes a substrate for the sulfate reduction pathway, whereas 4-hydroxy-benzoate (a�er further metabolic transformations) is assimilated as acetate.

Figure 1 .
Figure 1.Simplified scheme of the probable path of monoaryls biodegradation by sulfate-reducing bacteria.

Figure 2 .
Figure 2. Growh rate and H 2 S level during cultivation of Desulfotomaculum acetoxidans.1,Growth rate in medium with sulfate and lactate; 2, growth rate with 5-sulfo-salicylate; 3, H 2 S level in medium with sulfate and lactate; 4, H 2 S level in medium with 5-sulfo-salicylate.

Figure 7 .
Figure 7. Absorption spectrum of Pipes buffer extract of 1-day culture treated with 10 5 U lysozyme.

Figure 10 .
Figure 10.Proposed transformation reactions of secreted benzoate for following sulfate uptake processes.