A high-molecular-weight c-type cytochrome, Cyc2, and a putative 22-kDa c-type cytochrome were detected in the membrane fraction released during spheroplast formation from Acidithiobacillus ferrooxidans. This fraction was enriched in outer membrane components and devoid of cytoplasmic membrane markers. The genetics, as well as the subcellular localization of Cyc2 at the outer membrane level, therefore make it a prime candidate for the initial electron acceptor in the respiratory pathway between ferrous iron and oxygen.

An autotrophic bacterium able to gain energy from the oxidation of arsenite was isolated from arsenite-containing acid mine drainage waters. It belongs to the genus Thiomonas as shown by DNA-DNA hybridization experiments, 16S rRNA gene sequence, quinone and fatty acid content analyses. Carboxysomes were observed and the cbbSL genes encoding the ribulose 1,5-bisphosphate carboxylase/oxygenase were detected, confirming that this bacterium is able to fix CO(2). Arsenite oxidation was catalysed by a membrane-bound enzyme, and this activity was detected essentially in cells grown in the presence of arsenite. The genes encoding the two subunits of the arsenite oxidase of the Thiomonas isolate have been sequenced. The small subunit has a characteristic Tat signal sequence and contains the residues binding the [2Fe-2S] Rieske-type cluster. The large subunit has the [3Fe-4S] cluster-binding motif as well as the residues proposed to bind arsenite. In addition, most of the residues interacting with the molybdenum cofactor are conserved. The genes encoding both subunits belong to an operon, likely with a gene encoding a cytochrome c. The expression of this operon is greater in cells grown in the presence than in the absence of arsenite, in agreement with a transcriptional regulation in the presence of this metalloid.

The regulation of the expression of the rus operon, proposed to encode an electron transfer chain from the outer to the inner membrane in the obligate acidophilic chemolithoautroph Acidithiobacillus ferrooxidans, has been studied at the RNA and protein levels. As observed by Northern hybridization, real-time PCR and reverse transcription analyses, this operon was more highly expressed in ferrous iron- than in sulfur-grown cells. Furthermore, it was shown by immunodetection that components of this respiratory chain are synthesized in ferrous iron- rather than in sulfur-growth conditions. Nonetheless, weak transcription and translation products of the rus operon were detected in sulfur-grown cells at the early exponential phase. The results strongly support the notion that rus-operon expression is induced by ferrous iron, in agreement with the involvement of the rus-operon-encoded products in the oxidation of ferrous iron, and that ferrous iron is used in preference to sulfur.

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Despite the importance of Thiobacillus ferrooxidans in bioremediation and bioleaching, little is known about the genes encoding electron transfer proteins implicated in its energetic metabolism. This paper reports the sequences of the fourcox genes encoding the subunits of anaa 3-type cytochrome c oxidase. These genes are in a locus containing four other genes:cyc2, which encodes a high-molecular-weight cytochromec; cyc1, which encodes ac 4-type cytochrome (c 552); open reading frame 1, which encodes a putative periplasmic protein of unknown function; and rus, which encodes rusticyanin. The results of Northern and reverse transcription-PCR analyses indicated that these eight genes are cotranscribed. Two transcriptional start sites were identified for this operon. Upstream from each of the start sites was a ς70-type promoter recognized in Escherichia coli. While transcription in sulfur-grown T. ferrooxidans cells was detected from the two promoters, transcription in ferrous-iron-grown T. ferrooxidans cells was detected only from the downstream promoter. The cotranscription of seven genes encoding redox proteins suggests that all these proteins are involved in the same electron transfer chain; a model taking into account the biochemistry and the genetic data is discussed.

Introduction of chlA, B or E mutant alleles into strains carrying fusions between the lac structural genes and the promoter of the nitrate reductase operon led to the partial or total constitutive expression of the fusion. Presence of chlD mutated alleles in the same strains did not result in constitutive expression of the fusion and allowed full induction by nitrate only in the presence of molybdenum. It is proposed that the molybdenum cofactor, Mo-X, of the nitrate reductase is also corepressor of the operon. The chlA, B and E genes would be involved in the biosynthesis of the X-moity. Mutations in these genes would give an altered X-moity which still binds to molybdenum but leads to a less efficient repressor complex; chlD gene would code for an enzyme inserting molybdenum in the X-moity of the cofactor. Mutations in chlD give an empty cofactor leading to a complex which permanently represses the operon unless molybdenum is added.