Characterization of the petI and res Operons of Acidithiobacillus ferrooxidans

Abstract

DNA sequence analysis and bioinformatic interpretations have identified two adjacent clusters of genes potentially involved in the formation of a bc1 complex and in the maturation of a cytochrome c-type protein in two strains (ATCC 19859 and ATCC 33020) of the acidophilic, chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans (formerly Thiobacillus ferrooxidans). Reverse transcriptase-PCR experiments suggest that the two clusters are organized as operons, and ؉1 start sites of transcription for the operons have been determined by primer extension experiments. Potential promoters have been identified. The presence of these operons lends support to a recent model of reverse electron flow and is consistent with previous reports of phenotypic switching in this bacterium.Acidithiobacillus ferrooxidans, formerly Thiobacillus ferrooxidans (13), is a chemolithoautotrophic gram-negative bacterium that derives energy and electrons from the oxidation of ferrous iron and/or sulfur and various reduced sulfur compounds at pH 2 by using oxygen as the electron acceptor (11). It fixes CO 2 by the Calvin-Bassham-Benson scheme and can also fix nitrogen under microaerophilic conditions. A. ferrooxidans has been reported to be able to grow on hydrogen as an energy source both aerobically (4) and anaerobically by the dissimilatory reduction of Fe(III) (14). It has been shown to be active in the solubilization of copper and in the processing of refractory gold ores in bioleaching operations and is also a contributor to acid mine drainage.An intriguing problem is how A. ferrooxidans can generate the reduced pyridine nucleotides NADH and NADPH when using Fe(II) as the sole source of electrons and energy. The problem is that the standard reduction half-potential of the Fe 2ϩ /Fe 3ϩ couple (pH 2) is ϩ0.77 V while that of the NAD ϩ / NADH couple (pH 7) is Ϫ0.32 V. Therefore, to generate NADH, it is necessary to "push" electrons "uphill" from Fe(II) to NAD ϩ against a thermodynamically favorable gradient. It has been speculated that the energy required for this uphill electron flow could come primarily from the proton motive force resulting from an influx of protons across the A. ferrooxidans membrane since the exterior pH of the medium is 2 and that of the interior of the cell is 6.5 (12). It was further speculated that the pathway that the electrons take during uphill flow is from Fe(II) via a bc1 complex and a membranesoluble quinone to a NAD ϩ reductase complex (12). A downhill pathway of electrons in A. ferrooxidans has been described; in this pathway electrons derived from the oxidation of Fe(II) reduce oxygen to water via a series of electron carriers that remain inconclusively identified but that may include an outer membrane cytochrome (19), an iron-sulfur protein, blue copper protein rusticyanin, a cytochrome c 4 , and a terminal cytochrome aa 3 oxidase (1). This is considered the downhill pathway because electrons flow from the reduction potential of Fe 2ϩ (ϩ0.77 V) to the more positive reduction potential of O 2 , which...

Periplasmic Proteins of the Extremophile Acidithiobacillus ferrooxidans

“…4). It is known that cycA1 is codified by the petI operon and the cycA2 is in petII operon (65). Very recently, a model has been presented in which petI was proposed to encode the bc 1 complex, functioning in the uphill flow of electrons from iron to NAD(P), whereas petII was suggested to be involved in electron transfer from sulfur to oxygen (66).…”

Identification of a Gene Cluster for the Formation of Extracellular Polysaccharide Precursors in the Chemolithoautotroph Acidithiobacillus ferrooxidans

“…Several genes have been demonstrated to be differentially expressed in A. ferrooxidans grown in either iron or sulfur (16). With the goal of determining whether the oxidizable energy source also influenced the expression of genes potentially involved in galactose metabolism, a comparison was made by semiquantitative PCR of the expression of genes in the gal operon when A. ferrooxidans was grown in the presence of iron or sulfur.…”

Cytochromes c of Acidithiobacillus ferrooxidans

“…It has to be pointed out that, so far, A. ferrooxidans is the only organism characterized to contain two c 1 -type cytochromes. Four cytochromes belonged to the c 4 -type cytochrome family (24 925, 23 783, 21 171 and 20 768 Da), one of them being encoded by an orthologue of the A. ferrooxidans ATCC 33020 cyc1 gene [13], and another by an orthologue of the A. ferrooxidans ATCC 19859 cycA gene [15]. To our knowledge, A. ferrooxidans is the ¢rst microorganism found to contain genes encoding multiple c 4 -type cytochromes.…”