Characterization of an operon encoding two c-type cytochromes, an aa(3)-type cytochrome oxidase, and rusticyanin in Thiobacillus ferrooxidans ATCC 33020

Abstract

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 four cox genes encoding the subunits of an aa(3)-type cytochrome c oxidase. These genes are in a locus containing four other genes: cyc2, which encodes a high-molecular-weight cytochrome c; cyc1, which encodes a c(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 sigma70-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.

FIG. 1

RT-PCR on T. ferrooxidans total RNA. (A) Localizations of the oligonucleotides used for RT-PCR experiments; (B) RT-PCR experiments with oligonucleotides a and b on no template (lane 0), T. ferrooxidans genomic DNA (lane D), total RNA from T. ferrooxidans cells (lane R), total RNA from T. ferrooxidans but without reverse transcriptase (lane −); (C) RT-PCR experiments with oligonucleotides c and i, d and i, and f and i on RNA extracted from sulfur-grown cells (lanes R and −); (D) RT-PCR experiments with oligonucleotides e and i, f and i, and g and j on RNA extracted from ferrous-iron-grown cells (lanes R and −). Lanes M, 1-kb molecular weight ladder from Boehringer Mannheim.

FIG. 2

Northern blot of total RNA from ferrous-iron-grown T. ferrooxidans cells (1.1 μg) probed with DIG-UTP-labeled coxB RNA. The positions and the sizes of the RNA ladder from Gibco BRL are indicated on the left.

FIG. 3

Primer extension experiments. (A) Localizations of the oligonucleotides used. (B) RT experiments with the k, l, m, and n oligonucleotides on no template (lanes 0); E. coli carrying plasmid 1, 2, or 3 (lanes 1 to 3, respectively); total RNA from ferrous-iron (lanes F)- or sulfur (lanes S)-grown T. ferrooxidans (T.f) cells. Lanes M, [γ-³²P]ATP-labeled φX174 HinfI markers (from Promega).

FIG. 4

Proposed electron transfer pathway between ferrous iron and oxygen in T. ferrooxidans ATCC 33020. OM, outer membrane; IM, inner membrane; Cyt c4, cytochrome c₄; Hmc, high-molecular-weight cytochrome c.