Adenosine-5′-Phosphosulfate- and Sulfite Reductases Activities of Sulfate-Reducing Bacteria from Various Environments

A comparative study of the kinetic characteristics (specific activity, initial and maximum rate, and affinity for substrates) of key enzymes of assimilatory sulfate reduction (APS reductase and dissimilatory sulfite reductase) in cell-free extracts of sulphate-reducing bacteria (SRB) from various biotopes was performed. The material for the study represented different strains of SRB from various ecotopes. Microbiological (isolation and cultivation), biochemical (free cell extract preparation) and chemical (enzyme activity determination) methods served in defining kinetic characteristics of SRB enzymes. The determined affinity data for substrates (i.e., sulfite) were 10 times higher for SRB strains isolated from environmental (soil) ecotopes than for strains from the human intestine. The maximum rate of APS reductase reached 0.282–0.862 µmol/min×mg−1 of protein that is only 10 to 28% higher than similar initial values. The maximum rate of sulfite reductase for corrosive relevant collection strains and SRB strains isolated from heating systems were increased by 3 to 10 times. A completely different picture was found for the intestinal SRB Vmax in the strains Desulfovibrio piger Vib-7 (0.67 µmol/min × mg−1 protein) and Desulfomicrobium orale Rod-9 (0.45 µmol/min × mg−1 protein). The determinant in the cluster distribution of SRB strains is the activity of the terminal enzyme of dissimilatory sulfate reduction—sulfite reductase, but not APS reductase. The data obtained from the activity of sulfate reduction enzymes indicated the adaptive plasticity of SRB strains that is manifested in the change in enzymatic activity.


Introduction
Sulfate-reducing bacteria (SRB) are known to be the main producers of the biogenic hydrogen sulfide in the biosphere. They can be found in a number of natural ecotopes and in human-caused and Cultivation of SRB was performed in liquid modified Postgate C medium [40]. The highest sulfate concentration from 7.2 to 22.69 mmol L −1 was used in this modified Postgate C medium [31]. To adjust the pH (7.2-7.5), a sterile 10 mol L −1 solution of NaOH was used. The redox and anaerobic conditions were controlled by resazurin sodium (Oxoid, BR 0055B). Low redox potential (Eh = −100 ...−200 mV) for anaerobic condition was achieved by the addition of 2% ascorbic acid or 2% solution of sodium sulfide (1 mL L −1 of cultivation media). The tubes were filled with media, which were inoculated with SRB cultures (5% v/v), then closed by rubber plug to provide anaerobic conditions. The corrosive and soil SRB were cultivated at +28 • C for 7 days, while the intestinal bacteria were grown at +37 • C for 3 days.

Sulfate Determination
The content of sulfate in the medium was determined by the turbidimetric method right after the seeding and after 24 h of cultivation. Its essence lies in the precipitation of sulfate ions with the BaCl 2 and turbidimetric determination of it in the form of BaSO 4 . For the suspension stabilization, glycerol was used. Suspension of 40 mg L −1 BaCl 2 has been prepared in 0.12 mol L −1 HCl. The resulting solution was mixed with glycerol in a 1:1 ratio. To the 1 mL of sample supernatant after centrifugation at 5000× g at 23 • C (Hettich EBA 12 Centrifuge, Vlotho, Germany) was added 10 mL of prepared BaCl 2 :glycerol solution (in 1:1 ratio) and carefully stirred. The mixture was allowed to stand for 10 min and right after that the absorbance was measured at 520 nm (Spectrasonic Genesis 5, Berlin, Germany). As a control, the measurement was repeated with the same method using a cultivation medium. The calibration curve has been constructed with the same process. Calibration solutions have been prepared in distilled water at concentrations of 2,4,8,16,24,32,40, and 48 µmol L −1 of sodium sulfate [43].

Measurement of Hydrogen Sulfide
Production of hydrogen sulfide by SRB has been measured right after inoculating and after 24 h of cultivation spectrophotometrically. A 1 mL sample was added to 10 mL of a 5 g L −1 aqueous solution of zinc acetate. Right after this, 2 mL of a 0.75 g mL −1 of p-amino dimethylaniline in solution of sulfuric acid (2 mol L −1 ) was added. The mixture was left to stand for 5 min at room temperature. After that, 0.5 mL of 12 g L −1 solution of ferric chloride dissolved in 15 mmol L −1 sulfuric acid was added. After standing for another 5 min at room temperature, the mixture was centrifuged at 5000× g at 23 • C (Hettich EBA 12 Centrifuge, Vlotho, Germany). The absorbance of the centrifuged supernatant was determined to measure sulfide ions at a wavelength of 665 nm by spectrophotometer (Spectrasonic Genesis 5, Berlin, Germany). As a control, the measurement was repeated in the same method using a cultivation medium. The calibration curve was constructed with the same process. Calibration solutions were prepared in distilled water at concentrations of a 12.5, 25, 50, and 100 µmol L −1 of sodium sulfide [44].

Cell-Free Extracts
Cell-free extracts were prepared from the bacterial cell gained from the exponential phase of growth (1 day for intestinal strains, 7 days for corrosive-relevant strains). The bacteria were grown anaerobically in modified Postgate C liquid medium [31]. The cold extraction buffer (5 mol L −1 EDTA, 50 mmol L −1 potassium phosphate buffer, pH = 7.5) was added to centrifuged sediment cells to bind and depose heavy metal ions. After this procedure, the suspended bacterial cells containing 0.096-0.927 mg of protein × mL −1 were obtained. The cells were homogenized using the ultrasonic homogenizer (Bandelin SONOPULS GM 200, Berlin, Germany) at 20 kHz for 5 min at 0 • C. The soluble fractions were placed into centrifugal tubes and cell-free extracts were separated from the cell fragments by centrifugation for 30 min at 14,000× g and at 4 • C (Hettich EBA 12 Centrifuge, Vlotho, Germany). Supernatant was then used as cell-free extracts. The protein concentration in the cell-free extracts was determined by the Bradford method (1976) [45].

Kinetic Analysis
The study of kinetic properties of enzymes the APS reductase and sulfite reductase was performed as it described above. All experiments used to study the properties of these enzymes were performed using the initial rate V 0 (linear accumulation of product (P) in time). The kinetic parameters that characterized APS reductase, and sulfite reductase such as Michaelis constant (K m APS , K m Sulfite ) and maximum rate of these reactions (V max ) were determined by the Lineweaver-Burk plots [48]. The obtained concentration dependence of the rate of enzymatic reaction on the studied reagent (SO 3 2− ) was constructed in the coordinates (1/V on 1/S), where S is the concentration of the reagent (SO 3 2− ), and V is the rate of enzymatic oxidation of SO 3 2− at a concentration of SO 3 2− for APS reductase, reduction of SO 3 2− at a concentration of SO 3 2− for sulfite reductase, respectively (Tables S1-S5).

Statistical Analysis
Statistical calculations of the results were carried out using the MS Office (2010), Origin 8.0 (https://www.originlab.com/) and Statistica 13 (http://www.statsoft.com/) software programs. Cluster analysis was performed by the single linkage method with the calculation of the Euclidean distances. Using the experimental data, the basic statistical parameters (mean: M, standard error: m, M ± m) were calculated. The research results were treated by methods of variation statistics using Student's t-test. The significance of the calculated indicators of the line was tested by Fisher's F-test. The approximation was accurate when p ≤ 0.05 [49]. The cluster analyses were conducted by the inclusion of the following parameters: enzymes' specific activity, initial-maximum enzymatic reactions rates, and Michaelis constant. Statistical significance was also measured with the use of principal component analysis (PCA) that gave overall differences among compared groups. Statistical significance was also measured with the use of principal component analysis (PCA) by the inclusion of the same parameters as for the cluster analysis. PCA gave overall differences among compared groups.

Results
Two main metabolites of dissimilatory sulfate reduction processes were measured for studying SRB stains, isolated from various environments ( Table 2). Table 2. Hydrogen sulfide production during accumulation of sulfates by SRB cultures from various ecotopes (M ± m, n = 3; where "M" is average, "m" is mean, and "n" is the number of repetitions). The initial concentration of sulfate ions in the nutrient medium was in the range of the following values: 3.53-7.27 mmol L −1 . Initial concentrations of the sulfide-ion were from 0.20 to 1.01 mmol L −1 . After 24 h of SRB cultivation, the final concentrations of sulfate and sulfide ions in the culture medium were in the range 0.83-1.58 mmol L −1 and 1.19-4.86 mmol L −1 , respectively. The percentage ratio of consumed SO 4 2− and produced S 2− are shown in Figure 1. It was shown that in cultural media, the concentration of sulfate-ions decreased intensively by 62 to 88% (v/v), for all the SRB strains. Conversely, the production of sulfide ions for all SRB strains was increased by 26 to 52% (v/v). Desulfotomaculum sp. TC3 strain consumed the highest amount of SO4 2− (88% (v/v)) in comparison with other genera. Furthermore, the high consumption activity was shown by Desulfovibrio sp. TC2 (84% (v/v)), Desulfovibrio sp. 10 (84% (v/v)). The lowest consumption of sulfate ions was observed for the intestinal strains of D. orale Rod-9 and D. piger Vib-7; the reduction of sulfate ions in the culture medium was within the range: 62% (v/v) and 64% (v/v), respectively.
Among the tested strains, the maximum production of sulfide ions was revealed by bacteria isolated from the same ecotope-city heat system-Desulfovibrio sp. TC2 (52%), Desulfotomaculum sp. TC3. The lowest percentage (26% (v/v)) of sulfate was consumed by D. vulgaris DSM644. The increase in the production of sulfide ions (26 to 34%) by the intestinal strains of Rod-9 and Vib-7 was similar to that of D. vulgaris DSM644 and Desulfovibrio sp. 10 (26 to 30%).
It has to be emphasized that the production of sulfide ions did not correlate with the consumption of sulfate ions. The process of sulfate reduction may reflect the functional differences between SRB strains isolated from different habitats. Therefore, the kinetic parameters of key enzymes, including APS reductase and sulfite reductase, were subsequently analyzed.
Activated sulfate as APS is further reduced to sulfite by APS reductase and least to hydrogen sulfide by sulfite reductase [1,28]. These enzymes of dissimilatory sulfate reduction and their specific activities and kinetic parameters were calculated (Table 3, Figure 2). The specific activity data for both enzymes were different for SRB strains from different ecotopes. Activity values for APS reductase and sulfite reductase were in the range of 0.113-0.340 and 0.028-0.516 U/mL (0.8333-5.666 and 0.466-8.600 nkat), respectively. The highest activity of APS reductase was detected in cell-free extracts of the following strains: D. piger Vib-7 (5.666 nkat), It was shown that in cultural media, the concentration of sulfate-ions decreased intensively by 62 to 88% (v/v), for all the SRB strains. Conversely, the production of sulfide ions for all SRB strains was increased by 26 to 52% (v/v). Desulfotomaculum sp. TC3 strain consumed the highest amount of SO 4 2− (88% (v/v)) in comparison with other genera. Furthermore, the high consumption activity was shown by Desulfovibrio sp. TC2 (84% (v/v)), Desulfovibrio sp. 10 (84% (v/v)). The lowest consumption of sulfate ions was observed for the intestinal strains of D. orale Rod-9 and D. piger Vib-7; the reduction of sulfate ions in the culture medium was within the range: 62% (v/v) and 64% (v/v), respectively.
Among the tested strains, the maximum production of sulfide ions was revealed by bacteria isolated from the same ecotope-city heat system-Desulfovibrio sp. TC2 (52%), Desulfotomaculum sp. TC3. The lowest percentage (26% (v/v)) of sulfate was consumed by D. vulgaris DSM644. The increase in the production of sulfide ions (26 to 34%) by the intestinal strains of Rod-9 and Vib-7 was similar to that of D. vulgaris DSM644 and Desulfovibrio sp. 10 (26 to 30%).
It has to be emphasized that the production of sulfide ions did not correlate with the consumption of sulfate ions. The process of sulfate reduction may reflect the functional differences between SRB strains isolated from different habitats. Therefore, the kinetic parameters of key enzymes, including APS reductase and sulfite reductase, were subsequently analyzed.
Activated sulfate as APS is further reduced to sulfite by APS reductase and least to hydrogen sulfide by sulfite reductase [1,28]. These enzymes of dissimilatory sulfate reduction and their specific activities and kinetic parameters were calculated (Table 3, Figure 2). Table 3. Kinetics parameters of key enzymes of the dissimilatory sulfate-reduction pathway (M ± m, n = 3).

Sample
Specific Activity (nkat) Desulfovibrio sp. 10 (2.42 mmol L ). For the intestinal D. piger Vib-7 strain, it is shown that the high specific activity of APS reductase (5.666 ± 0.483 nkat) coincides with the affinity for the substrate, expressed by the Michaelis constant. Although D. vulgaris DSM644 had the higher specific activity of APS reductase, the value of Km APS (0.99 mmol L −1 ) was the lowest Km APS (0.99 mM) among the studied strains. Rod-9 has the lowest specific activity of APS reductase, but a significant affinity to APS substrate (Km APS = 3.57 mmol L −1 ). The specific activity of APS reductase for SRB strains isolated from different ecotopes did not match the affinity for the substrate.  Rod-9 has the lowest specific activity of APS reductase, but a significant affinity to APS substrate (K m APS = 3.57 mmol L −1 ). The specific activity of APS reductase for SRB strains isolated from different ecotopes did not match the affinity for the substrate. The data of activity of the enzyme in the final step of the sulfate reduction process corresponded to the calculated Michaelis constant values, and the affinity for the substrate (sulfite) was the highest in Desulfovibrio sp. 10 (46.73 mmol L −1 ), Desulfotomaculum sp. TC3 (46.17 mmol L −1 ) and D. vulgaris DSM644 (31.67 mmol L −1 ). It is characteristic that the smallest specific activity of sulfite reductase was detected in intestinal strains of SRB (0.466-0.533 nkat) and the least affinity for the substrate was determined according to the data of the Michaelis constant (3.53-3.86 mmol L −1 ). Therefore, the specific sulfite reductase activity for all tested SRB strains isolated from different ecotopes practically coincided with the affinity for the substrate.

APS
Important indicators of the reaction kinetics involved in the processes of dissimilatory sulfate reduction are the rates of product conversion reactions. The initial (V 0 ) and maximum (limiting) V max rates of enzymatic reactions were determined (Figure 3). The SRB strains have an initial rate of V 0 of substrate consumption for APS reductase of 0.231-0.675 µmol/min × mg −1 protein. The highest initial velocity values of this enzyme were found in D. vulgaris strain DSM644 (0.639 µmol/min × mg −1 protein) and D. piger Vib-7 (0.675 µmol/min × mg −1 protein). The maximum rate of APS reductase reached 0.282-0.862 µmol/min × mg −1 of protein, which is only 10 to 28% higher than similar initial values. For sulfite reductase, V0 was 0.049-0.076 µmol/min × mg −1 protein for corrosion-active strains isolated from collection and the strains isolated from heating systems, and the initial intestinal SRB rate was quite different (0.138 and 0.351 µmol/min × mg −1 protein for D. piger Vib-7 and D.orale Rod-9, respectively). It is significantly higher by 2.81-4.61 times than the minimum initial velocity of 0.049 µmol/min×mg −1 protein for DVI-10 strain. The maximum rate of sulfite reductase reached values of 0.23-0.516 µmol/min×mg −1 protein (for corrosion-active collection strains and SRB strains isolated from heating systems). The rate increased by 2.98-10.32 times. However, a completely different picture was found for intestinal SRB, where the Vmax in strains for D. piger Vib-7 (0.067 µmol/min × mg −1 protein) and D. orale Rod-9 (0.045 µmol/min×mg −1 protein) was decreased significantly by 5.24 and 3.06 times, respectively.
To analyze the affinity of SRB strains according to the kinetic parameters of two enzymes of dissimilatory sulfate reduction, a cluster analysis was performed. Figure 4 shows the cluster analysis results. The results indicate differences among evaluated bacterial strains toward applied enzymes (APS reductase, sulfite reductase, and both enzymes together). The APS reductase parameter divided the studied strains into twoclusters. The first cluster was divided into three sub-clusters: the first TC4 and DSM642; the second DVI-10 and TC2; the third DSM644 and TC3. The intestinal strain ROD-9 joined a cluster, but divides from sub-clusters. In addition, SRB Vib-7 was separated and did not belong to any cluster. Sulfite reductase parameters formed following separated clusters: the first cluster contained VIB-7 and ROD-9 with additional strain DSM642; the second cluster contained DSM644, TC2 and TC4; and the third cluster contained DVI-10 and TC3. Almost the same results (as sulfite reductase) were obtained when both parameters were included in one cluster analysis ( Figure  4). Therefore, these results indicate that the determinant in the cluster distribution of strains of SRB is the activity of the terminal enzyme of dissimilatory sulfate reduction-sulfite reductase, not APS reductase. This difference can be caused by the presence of SRBs in certain environments (soils, corrosion products or human intestine), which are selective for intestinal SRB strains. For sulfite reductase, V 0 was 0.049-0.076 µmol/min × mg −1 protein for corrosion-active strains isolated from collection and the strains isolated from heating systems, and the initial intestinal SRB rate was quite different (0.138 and 0.351 µmol/min × mg −1 protein for D. piger Vib-7 and D.orale Rod-9, respectively). It is significantly higher by 2.81-4.61 times than the minimum initial velocity of 0.049 µmol/min×mg −1 protein for DVI-10 strain. The maximum rate of sulfite reductase reached values of 0.23-0.516 µmol/min×mg −1 protein (for corrosion-active collection strains and SRB strains isolated from heating systems). The rate increased by 2.98-10.32 times. However, a completely different picture was found for intestinal SRB, where the V max in strains for D. piger Vib-7 (0.067 µmol/min × mg −1 protein) and D. orale Rod-9 (0.045 µmol/min×mg −1 protein) was decreased significantly by 5.24 and 3.06 times, respectively.
To analyze the affinity of SRB strains according to the kinetic parameters of two enzymes of dissimilatory sulfate reduction, a cluster analysis was performed. Figure 4 shows the cluster analysis results. The results indicate differences among evaluated bacterial strains toward applied enzymes (APS reductase, sulfite reductase, and both enzymes together). The APS reductase parameter divided the studied strains into twoclusters. The first cluster was divided into three sub-clusters: the first TC4 and DSM642; the second DVI-10 and TC2; the third DSM644 and TC3. The intestinal strain ROD-9 joined a cluster, but divides from sub-clusters. In addition, SRB Vib-7 was separated and did not belong to any cluster. Sulfite reductase parameters formed following separated clusters: the first cluster contained VIB-7 and ROD-9 with additional strain DSM642; the second cluster contained DSM644, TC2 and TC4; and the third cluster contained DVI-10 and TC3. Almost the same results (as sulfite reductase) were obtained when both parameters were included in one cluster analysis (Figure 4). Therefore, these results indicate that the determinant in the cluster distribution of strains of SRB is the activity of the terminal enzyme of dissimilatory sulfate reduction-sulfite reductase, not APS reductase. This difference can be caused by the presence of SRBs in certain environments (soils, corrosion products or human intestine), which are selective for intestinal SRB strains. Principal component analysis (PCA) was used to separate SRB strains (TC3, TC4, DVI-10, DSM642, VIB-7, TC2, DSM644 and ROD-9) according to their parameters of APS and sulfite reductases separately and together ( Figure 5). The following strains, TC3, TC4, DVI-10, DSM642, VIB-7 and TC2, formed one cluster by the parameters of APS reductase. DSM644 and ROD-9 were separated from this cluster. Conversely, no  Principal component analysis (PCA) was used to separate SRB strains (TC3, TC4, DVI-10, DSM642, VIB-7, TC2, DSM644 and ROD-9) according to their parameters of APS and sulfite reductases separately and together ( Figure 5). The following strains, TC3, TC4, DVI-10, DSM642, VIB-7 and TC2, formed one cluster by the parameters of APS reductase. DSM644 and ROD-9 were separated from this cluster. Conversely, no The following strains, TC3, TC4, DVI-10, DSM642, VIB-7 and TC2, formed one cluster by the parameters of APS reductase. DSM644 and ROD-9 were separated from this cluster. Conversely, no differences were observed between SRB strains in sulfite reductase parameters. Based on the activity of both enzymes (APS and sulfite reductase), VIB-7 and ROD-9 were separated from the rest of the SRB.

Discussion
The sulfate reduction enzymes are located in the cytoplasm and peripheral plasma. The sulfate ions can be transported into the cells simultaneously with protons and some sulfate-reducing bacteria, absorbing sulfate from the flow of sodium ions [1]. This high activity in the cell-free extracts may be caused by determining it in a crude sample of intestinal SRB grown only in a selective medium for SRB. On the other hand, it can also be based on their inhabitant location, which is the large intestine of rats and mice [22,23].
The second key enzyme, APS reductase, is mainly found in the dissimilatory SRB, such as species of Desulfovibrio and Desulfotomaculum genera, and in some oxidizing sulfite to sulfate Thiobacillus spp. [50]. The specific enzymatic activity (0.095 U × mg of protein −1 ) of this enzyme was assayed by Huisingh et al. (1974) [46] for a Desulfovibrio sp. strain isolated from sheep rumen and described. Similar activity was also observed in a crude cell-free extract of D. desulfuricans (0.08 U × mg of protein −1 ) [50]. Our obtained data about specific activity values for APS reductase were in the range 0.113-0.340 U/mL (0.8333-5.666 nkat). It can be seen that the specific activity of APS reductase in cell-free extracts of the strains was two orders of magnitude higher than in the literature. Enzymatic mechanism of sulfite reduction in SRB isolated from the environment was elucidated by Isimoto and Yagi (1960) and Seki et al. (1978) [51,52]. The specific activity of sulfite reductase from D. vulgaris was determined to be 0.19 U × mg of protein −1 in both fractions after DEAE-Sephadex column chromatography [53]. On the other hand, higher activity (0.99 U × mg of protein −1 ) of this enzyme was detected in supernatants of crude extracts from Desulfovibrio spp. [50]. The specific enzymatic activities obtained from our studied SRB samples ranging from 0.028 ± 0.001 to 0.516 ± 0.04 U × mg of protein −1 were in the range of activities mentioned above. Intestinal SRB isolated from the large intestine of rodents (rats and mice) show differences in enzymatic activity for sulfite reductase in each sample taken (0.317 ± 0.037 to 0.702 ± 0.023 U × mg of protein −1 ), even in the similar group of SRB strains. The maximum rate of sulfite reductase in intestinal strains (Vib-7 and Rod-9) was lower than the initial rate in the corrosion-active SRB strains, in which the maximum rate was expected to be higher than the initial one (see Figure 3).
Generally, a reduction of sulfate became a dominant biological process in the water environment (oceans and deposits), resulting in sulfidic anoxic conditions from 2.5 to 0.6 billion years ago [54,55]. Currently, it is well known that SRB are able to colonize habitats that are different from the aquatic anaerobic environment where microorganisms have existed for a long time; in particular, the intestinal tract of animals (sea urchins, rodents, cows) and humans [56,57], where microorganisms need to adapt to new living conditions. The confirmation of this is that affinity data for substrates indicate that high affinity was shown for sulfite by SRB strains isolated from natural ecotopes, mainly in the soil environment. Additionally, the fact that the content of sulfates/sulfites in the aquatic environment (in particular in marine) (up to 2.7 g L −1 or soluble sulfate-ion 0.0283 moles kg −1 water) [3,58] differs from those present in the intestinal lumen and that come from food and beverages [59] may be a selective factor leading to changes in the functioning of the enzymatic system. Subseafloor microbial ecosystems transform sulfur compounds; approximately 11.3 moles of sulfate are microbially reduced each year, accounting for the oxidation of 12 to 29% of the organic carbon flux to the seafloor [3]. Dissimilatory sulfate reduction by marine sediments largely depends on the availability of sulfate supplied from seawater [60,61].
The sulfite reductases belong to a family of proteins that also include the assimilatory sulfite and nitrite reductases [54,62]. These enzymes reduce sulfite to a mixture of tritionate, thiosulfate and sulfide in proportions that depend on the reaction (environmental) conditions [63]. In some Desulfovibrio spp. strains, through the regulation of nitrate and nitrite oxidation, induction by nitrate and repression by sulfate is possible (i.e., sulfate reduction in the presence of nitrates is suppressed) [64]. It is known that the same reactions in SRB can work in energetic and constructive directions, that is, be reversible [54]. Extensive metabolic plasticity of SRB is making them capable to use other reduced sources of sulfur in the absence of sulfate. In addition, they are capable of performing nitrate reduction, nitrite reduction, acetogenic reactions and the transfer of molecular hydrogen to other microorganisms. All these qualities bring the SRB closer to other anaerobes and provide space for constructing evolutionary circuits and information transmission pathways between different anaerobic groups [28,[65][66][67]. As discussed in the studies by Klein (2001), similar patterns of insertions and deletions in ApsA sequences of donor and recipient lineages provide additional evidence for lateral gene transfer. From a subset of reference strains (n = 25), a fragment of the dissimilatory sulfite reductase genes (dsrAB) that have recently been proposed to have undergone multiple lateral gene transfers [68] was also amplified and sequenced. Phylogenetic comparison of DsrAB-and ApsA-based trees suggests a frequent involvement of Gram-positive and thermophilic SRB in lateral gene transfer events among the SRB. Further patterns of lateral gene transfer (LGTs) in SRB exist; these may also be linked to their ecophysiology. Most recipients of xenologous dsrAB and apsA genes are thermophilic [28]. We can assume that the likely intestinal SRB strains have acquired some adaptation to the conditions of the intestinal tract that is significantly different both in the content of sulfate and in elevated temperature from the conditions of the soil and water environments.
In view of the fact that Salmonella enterica [36] and Clostridium pasteurianum [37] utilize sulfate in the assimilatory sulfate reduction process and the main product is not hydrogen sulfide released from the outside, but rather is a cysteine, which is a part of sulfur-containing proteins. A dissimilatory sulfite reductase was purified from Bilophila wadsworthia RZATAU and is involved in energy conservation by reducing sulfite to sulfide, during the degradation of taurine as an electron acceptor [69]. This enzyme is also found in some phototrophic and chemotrophic sulfur oxidizers, where it is proposed to operate in the reverse direction (reverse sulfite reductase, rDSR) [54,70]. To confirm this, a recombinant Vib-7M strain was constructed that contained gene CysK coded O-acetyl-serine(thiol)lyase, which is an enzyme catalyzing the reaction of inorganic sulfide with O-acetyl-serine to form the S-containing amino acid l-cysteine [56]. Mutant strains are capable of higher speeds (1.5 times) using sulfate compared with the strains that are in the intestines of animals with colitis. Therefore, it can be assumed that, unlike strains isolated from the soil, including strains DVI-10, DSM642 and DSM644, for the intestinal strains Vib-7 and ROD-9, there is competition in trying to restructure the metabolism similar to that prevalent in the microorganisms of the intestinal microbiota. This is manifested in a decreased rate of sulfite reductase, and a decreased affinity to the substrate (sulfites). For intestinal strains of SRB, similar results were obtained for the first step of sulfate-reduction pathway, i.e., ATP sulfurylase. We discussed that this decrease in the maximum rate of the enzyme could show the inhibition of the enzymatic process of sulfate accumulation in the bacterial cells at the end of the reaction [39]. Therefore, it is possible that, in addition to competition for substrates with methanogens by the intestinal SRB strains, inhibition of the final stages of sulfate reduction may occur [71,72].
The cluster analysis data confirm changes in the adaptation of SRB strains to different existence conditions, and indicate that the terminal enzyme (sulfite reductase) is crucial in the adaptation of bacterial cells to environmental conditions, namely changes in their specific activity.

Conclusions
A comparative study of the kinetic characteristics (specific activity, maximum rate, and affinity of the substrates) of dissimilatory sulfate reduction key enzymes (in particular APS reductase and dissimilatory sulfite reductase) was carried out. Determined affinity data for substrates indicate a high affinity for sulfite by SRB strains isolated from natural ecotopes, mainly in the soil environment. The maximum rate of sulfite reductase in intestinal strains (Vib-7 and Rod-9) is lower than the initial rate, and also than the initial one in the corrosive relevant SRB strains. The determinant in the cluster distribution of SRB strains is the activity of the terminal enzyme of dissimilatory sulfate reduction-sulfite reductase, not APS reductase. This difference could be caused by the presence of sulfite-reducing bacteria in environments (soils, corrosion products or human intestine) in which one is selective for intestinal SRB but, without isolation, the contamination of sulfite-reducing bacteria can be expected.