Downloaded from www.microbiologyresearch.org by IP: 23.20.98.176 On: Tue, 05 Apr 2016 07:11:28 Respiratory gene clusters of Metallosphaera sedula – differential expression and transcriptional organization Ulrike Kappler, 1,2 Lindsay I. Sly 1 and Alastair G. McEwan 1,2 Correspondence Alastair G. McEwan mcewan@uq.edu.au School of Molecular and Microbial Sciences 1 and Centre for Metals in Biology 2 , The University of Queensland, St Lucia, Qld 4072, Australia Received 27 July 2004 Revised 17 September 2004 Accepted 5 October 2004 Metallosphaera sedula is a thermoacidophilic Crenarchaeon which is capable of leaching metals from sulfidic ores. The authors have investigated the presence and expression of genes encoding respiratory complexes in this organism when grown heterotrophically or chemolithotrophically on either sulfur or pyrite. The presence of three gene clusters, encoding two terminal oxidase complexes, the quinol oxidase SoxABCD and the SoxM oxidase supercomplex, and a gene cluster encoding a high-potential cytochrome b and components of a bc 1 complex analogue (cbsBA–soxL2N gene cluster) was established. Expression studies showed that the soxM gene was expressed to high levels during heterotrophic growth of M. sedula on yeast extract, while the soxABCD mRNA was most abundant in cells grown on sulfur. Reduced-minus-oxidized difference spectra of cell membranes showed cytochrome-related peaks that correspond to published spectra of Sulfolobus-type terminal oxidase complexes. In pyrite-grown cells, expression levels of the two monitored oxidase gene clusters were reduced by a factor of 10–12 relative to maximal expression levels, although spectra of membranes clearly contained oxidase-associated haems, suggesting the presence of additional gene clusters encoding terminal oxidases in M. sedula. Pyrite- and sulfur-grown cells contained high levels of the cbsA transcript, which encodes a membrane-bound cytochrome b with a possible role in iron oxidation or chemolithotrophy. The cbsA gene is not co-transcribed with the soxL2N genes, and therefore does not appear to be an integral part of this bc 1 complex analogue. The data show for the first time the differential expression of the Sulfolobus-type terminal oxidase gene clusters in a Crenarchaeon in response to changing growth modes. INTRODUCTION Sulfide forms highly insoluble minerals with a number of metals of major economic and industrial importance. Since the isolation of bacteria such as Acidithiobacillus (formerly Thiobacillus) ferrooxidans in the 1950s it has been established that micro-organisms greatly enhance the extraction of metals by leaching processes (reviewed by Rawlings, 2001). The predominantly accepted view of the role of micro-organisms in the solubilization of metal sulfides is that they provide protons from the oxidation of sulfur compounds and maintain iron in its oxidized form for Fe 3+ /H + attack on the mineral (Sand et al., 2001). Over three decades of research on mesophilic bacteria such as A. ferrooxidans has led to an understanding of the organi- zation of the electron transfer chain in this chemolitho- autotroph (Brasseur et al., 2002; Ohmura et al., 2002). In addition to the acidophilic bioleaching bacteria, a number of thermophilic Crenarchaeota are also able to oxidize sulfur compounds and sulfidic metal ores and can achieve leaching rates superior to those of mesophilic micro- organisms, making them attractive for use in commercial tank leaching operations (Dew et al., 2000). However, at present, our knowledge about the physiological features of these organisms which enable this enhanced performance is rather limited. The oxidation of ferrous iron and inorganic sulfur mole- cules is an aerobic process that is linked to the respiratory chain. Over the last decade or more there has been con- siderable progress towards an understanding of the mole- cular properties of the oxidases and associated electron transfer proteins in Crenarchaeota, especially Sulfolobus acidocaldarius (Pereira et al., 2004; Schafer et al., 1999; Schmidt, 2004). Biochemical and spectroscopic character- ization of respiratory complexes has been complemented by molecular genetic studies, and the latter have been greatly facilitated by the availability of sequenced genomes for Sulfolobus solfataricus and Sulfolobus tokodaii (Kawarabayasi Abbreviation: YE, yeast extract. The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AY452058, AY452059, AY452060 and AY452061. 0002-7515 G 2005 SGM Printed in Great Britain 35 Microbiology (2005), 151, 35–43 DOI 10.1099/mic.0.27515-0