Metagenomic and Biochemical Characterizations of Sulfur Oxidation Metabolism in Uncultured Large Sausage-Shaped Bacterium in Hot Spring Microbial Mats

<div><p>So-called “sulfur-turf” microbial mats in sulfide containing hot springs (55–70°C, pH 7.3–8.3) in Japan were dominated by a large sausage-shaped bacterium (LSSB) that is closely related to the genus <em>Sulfurihydrogenibium</em>. Several previous reports proposed that the LSSB would be involved in sulfide oxidation in hot spring. However, the LSSB has not been isolated yet, thus there has been no clear evidence showing whether it possesses any genes and enzymes responsible for sulfide oxidation. To verify this, we investigated sulfide oxidation potential in the LSSB using a metagenomic approach and subsequent biochemical analysis. Genome fragments of the LSSB (a total of 3.7 Mb sequence including overlapping fragments) were obtained from the metagenomic fosmid library constructed from genomic DNA of the sulfur-turf mats. The sequence annotation clearly revealed that the LSSB possesses sulfur oxidation-related genes coding sulfide dehydrogenase (SD), sulfide-quinone reductase and sulfite dehydrogenase. The gene encoding SD, the key enzyme for sulfide oxidation, was successfully cloned and heterologously expressed in <em>Escherichia coli</em>. The purified recombinant enzyme clearly showed SD activity with optimum temperature and pH of 60°C and 8.0, respectively, which were consistent with the environmental conditions in the hot spring where the sulfur-turf thrives. Furthermore, the affinity of SD to sulfide was relatively high, which also reflected the environment where the sulfide could be continuously supplied. This is the first report showing that the LSSB harbors sulfide oxidizing metabolism adapted to the hot spring environment and can be involved in sulfide oxidation in the sulfur-turf microbial mats.</p> </div

Comparisons of kinetic properties of recombinant DhsU1 of the LSSB with previously known SDs from other bacteria.

*<p>ND, not determined.</p

Conceptual model of inorganic sulfur compounds oxidation pathway and respiratory complex in the LSSB.

<p>Sulfide dehydrogenase, DhsU; sulfide-quinone reductase, Sqr; sulfite dehydrogenase, Sor; sox complex, SoxXYZAB; quinone, Q; cytochrome <i>c</i>, Cyt <i>c</i>; cytochrome <i>bc1</i> complex, Cyt <i>bc1</i> complex; <i>Cbb3</i>-type cytochrome <i>c</i> oxidase, <i>Cbb3</i>-type cyt <i>c</i> oxidase. The solid arrow shows the reaction confirmed by both metagenomic and biochemical approaches. The dashed arrows indicate the putative reactions identified by only metagenomic information.</p

Schematic map of the <i>dhsU</i> gene cluster found in the LSSB.

<p>Arrows indicate the position and direction of each ORF. Black, light-gray, dark-gray, and white arrows show <i>dhsU</i> genes, cytochrome-like genes, sox-related genes, and a Fe-S protein gene and a ferredoxin gene, respectively.</p

Photographs of sulfur-turf microbial mat.

<p>[A] A photograph of sulfur-turf microbial mat found in the hot spring stream, [B] a phase contrast photomicrograph of the LSSB with elemental sulfur granules. An arrow indicates flowing channel of sulfide-containing hot spring water. Scale bar = 10 µm.</p

Effect of [A] pH and [B] temperature on recombinant LSSB DhsU1 and [C] its thermostability.

<p>The optimal temperature was determined by measuring the enzyme activity at selected temperatures from 40–80°C. The optimal pH was determined in several buffers with a range from 7.0–10.0 (50 mM): Tris-HCl (7.0–9.0) and CAPS (9.5–10.0). The thermostability was determined by using pre-incubated DhsU1 for 30 min at 30–90°C.</p

Purification of recombinant DhsU1 of the LSSB.

<p>The reaction mixture contained 100 mM Tris-HCl (pH 8.0), 30 µM horse heart cytochrome <i>c</i>, 10 µM sodium sulfide and 25 µg crude or purified DhsU1. The total volume of the reaction mixture was 1.0 ml. The reaction was started by adding sodium sulfide. One unit of activity (U) is defined as 1 µmol of horse-heart cytochrome <i>c</i> reduced per minute.</p

Phylogenetic relationship of DhsU1 homologs including real and putative SD between LSSB and other bacteria.

<p>The tree was constructed using neighbour-joining method based on the amino acid sequences. The scale bar indicates 0.1 substitutions per amino acid position. The numbers at nodes represent bootstrap values (100 replicates). Asterisk indicates the real SDs previously proved to have its enzymatic activity <i>in vitro</i>.</p

Ultraviolet-visible absorption spectrum of recombinant DhsU1 of the LSSB.

<p>DhsU1 (4.5 mg/ml) in 100 mM Tris-HCl buffer (pH 8.0) at room temperature. (A) air-oxidized DhsU1, (B) dithionite-reduced DhsU1.</p

SDS-PAGE of recombinant DhsU1 of the LSSB.

<p>Lane 1, crude fraction; lane 2, purified DhsU1; M, molecular weight standard (19.5–104.3 kDa).</p