An Extracellular Tetrathionate Hydrolase from the Thermoacidophilic Archaeon Acidianus Ambivalens with an Activity Optimum at pH 1.

Fabian Müller,Arnulf Kletzin,Reinhard Mentele,Bastian Naß,Friedrich Lottspeich,Karin Lauber,Jonas Protze

doi:10.3389/fmicb.2011.00068

Abstract

Background: The thermoacidophilic and chemolithotrophic archaeon Acidianus ambivalens is routinely grown with sulfur and CO2-enriched air. We had described a membrane-bound, tetrathionate (TT) forming thiosulfate:quinone oxidoreductase. Here we describe the first TT hydrolase (TTH) from Archaea. Results: A. ambivalens cells grown aerobically with TT as sole sulfur source showed doubling times of 9 h and final cell densities of up to 8 × 108/ml. TTH activity (≈0.28 U/mg protein) was found in cell-free extracts of TT-grown but not of sulfur-grown cells. Differential fractionation of freshly harvested cells involving a pH shock showed that about 92% of the TTH activity was located in the pseudo-periplasmic fraction associated with the surface layer, while 7.3% and 0.3% were present in the soluble and membrane fractions, respectively. The enzyme was enriched 54-fold from the cytoplasmic fraction and 2.1-fold from the pseudo-periplasmic fraction. The molecular mass of the single subunit was 54 kDa. The optimal activity was at or above 95°C at pH 1. Neither PQQ nor divalent cations had a significant effect on activity. The gene (tth1) was identified following N-terminal sequencing of the protein. Northern hybridization showed that tth1 was transcribed in TT-grown cells in contrast to a second paralogous tth2 gene. The deduced amino acid sequences showed similarity to the TTH from Acidithiobacillus and other proteins from the PQQ dehydrogenase superfamily. It displayed a β-propeller structure when being modeled, however, important residues from the PQQ-binding site were absent. Conclusion: The soluble, extracellular, and acidophilic TTH identified in TT-grown A. ambivalens cells is essential for TT metabolism during growth but not for the downstream processing of the TQO reaction products in S°-grown cells. The liberation of TTH by pH shock from otherwise intact cells strongly supports the pseudo-periplasm hypothesis of the S-layer of Archaea.

Highlights

The dissimilatory oxidation of inorganic sulfur compounds (ISC) is one of the most important sources of metabolic energy for CO2 fixation in light-independent ecosystems like solfataras and other volcanic hot springs
There are many gaps in our knowledge concerning the mechanisms of ISC oxidation in acidophilic Bacteria and Archaea, the dominant microorganisms in volcanic hot springs and bioleaching environments (Kletzin, 2007, 2008; Ghosh and Dam, 2009)
Some final conclusions for the A. ambivalens sulfur metabolism can be drawn: One of the initial questions was, how TT produced in the thiosulfate:quinone oxidoreductase (TQO) reaction is metabolized (Müller et al, 2004)

Summary

Introduction

The dissimilatory oxidation of inorganic sulfur compounds (ISC) is one of the most important sources of metabolic energy for CO2 fixation in light-independent ecosystems like solfataras and other volcanic hot springs (reviewed for example by Kelly et al, 1997; Friedrich et al, 2005; Stetter, 2006; Kletzin, 2007; Ghosh and Dam, 2009). A soluble sulfur oxygenase reductase (SOR) mediates the initial step in the S° oxidation pathway of aerobically grown cells, a unique enzyme catalyzing the simultaneous S° oxygenation and disproportionation to sulfite, thiosulfate, and sulfide (Kletzin, 1989; Urich et al, 2004; Veith et al, 2011; this volume). Regardless of that, S°-grown A. ambivalens cells contain significant amounts of a membrane-bound thiosulfate:quinone oxidoreductase (TQO) oxidizing thiosulfate to tetrathionate (TT) and reducing quinones (Müller et al, 2004).

Methods

Results

Discussion

Conclusion