Acidithiobacillus ferrooxidans is an acidophilic chemolithoautotrophic bacterium that can grow in the presence of either the weak reductant Fe(2+), or reducing sulfur compounds that provide more energy for growth than Fe(2+). We have previously shown that the uphill electron transfer pathway between Fe(2+) and NAD(+) involved a bc(1) complex that functions only in the reverse direction [J. Bacteriol. 182, (2000) 3602]. In the present work, we demonstrate both the existence of a bc(1) complex functioning in the forward direction, expressed when the cells are grown on sulfur, and the presence of two terminal oxidases, a bd and a ba(3) type oxidase expressed more in sulfur than in iron-grown cells, besides the cytochrome aa(3) that was found to be expressed only in iron-grown cells. Sulfur-grown cells exhibit a branching point for electron flow at the level of the quinol pool leading on the one hand to a bd type oxidase, and on the other hand to a bc(1)-->ba(3) pathway. We have also demonstrated the presence in the genome of transcriptionally active genes potentially encoding the subunits of a bo(3) type oxidase. A scheme for the electron transfer chains has been established that shows the existence of multiple respiratory routes to a single electron acceptor O(2). Possible reasons for these apparently redundant pathways are discussed.
The nature of the mineral–bacteria interphase where electron and mass transfer processes occur is a key element of the bioleaching processes of sulfide minerals. This interphase is composed of proteins, metabolites, and other compounds embedded in extracellular polymeric substances mainly consisting of sugars and lipids (Gehrke et al., Appl Environ Microbiol 64(7):2743–2747, 1998). On this respect, despite Acidithiobacilli—a ubiquitous bacterial genera in bioleaching processes (Rawlings, Microb Cell Fact 4(1):13, 2005)—has long been recognized as secreting bacteria (Jones and Starkey, J Bacteriol 82:788–789, 1961; Schaeffer and Umbreit, J Bacteriol 85:492–493, 1963), few studies have been carried out in order to clarify the nature and the role of the secreted protein component: the secretome. This work characterizes for the first time the sulfur (meta)secretome of Acidithiobacillus thiooxidans strain DSM 17318 in pure and mixed cultures with Acidithiobacillus ferrooxidans DSM 16786, identifying the major component of these secreted fractions as a single lipoprotein named here as Licanantase. Bioleaching assays with the addition of Licanantase-enriched concentrated secretome fractions show that this newly found lipoprotein as an active protein additive exerts an increasing effect on chalcopyrite bioleaching rate.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-010-3063-8) contains supplementary material, which is available to authorized users.
Acidithiobacillus ferrooxidans, formerly Thiobacillus ferrooxidans (24), is a gram-negative bacterium that has been shown to be active in the solubilization of copper and in the processing of refractory gold ores in bioleaching operations (reviewed in references 21 and 36). It is also a major contributor to acid mine drainage in copper and coal mines and in certain natural environments. It is a chemolithotroph, deriving energy and electrons from the oxidation of ferrous iron and/or sulfur and various reduced sulfur compounds at pH 2 to 4, using oxygen as the ultimate electron acceptor (22). It fixes CO 2 by the Calvin-Bassham scheme. It can also anaerobically oxidize hydrogen at pH 5.5 (15). Recently, the almost complete genome sequence of A. ferrooxidans was used to detect and inventory the genes involved in amino acid metabolism (40).A mutant of A. ferrooxidans ATCC 19859 has been isolated that is able to switch reversibly, and with high frequency, between a wild-type state, in which it can oxidize both ferrous iron and sulfur compounds, and a mutant state, in which it has lost the capacity to oxidize iron (39). This phenomenon resembles other states of instability associated with the transposition of insertion sequences that have been described in other organisms and led us to investigate whether phenotypic switching might similarly be explained in A. ferrooxidans.Evidence was recently presented (5) that implicated a member of the so-called family 1 repetitive elements (50) in phenotypic switching. This repetitive element was tentatively identified as an insertion sequence and termed IST1 (renamed ISAfe1 here). Phenotypic switching was shown to be correlated with the high frequency insertion and excision of ISAfe1 into, and out of, the resB gene (5). ResB encodes a cytochrome c-type maturation protein (reviewed in reference 45), and a model was proposed in which insertion of ISAfe1 into resB eliminated the capacity of ResB to satisfactorily mature a c-type cytochrome and that this resulted, in turn, in the loss of the ability to oxidize iron but not sulfur (5).In order to describe and explain the phenomenon of phenotypic switching, we carried out a partial molecular characterization of ISAfe1. We further demonstrate that ISAfe1 can promote plasmid integration and resolution in E. coli, opening up the future possibility of exploiting E. coli to test experimentally certain characteristics of this insertion sequence. MATERIALS AND METHODSBacterial strains and media. Strains and plasmids used in this study are listed in Table 1. A. ferrooxidans ATCC 19859 was grown on Mackintosh medium or in modified 9K-ferrous iron medium (50). E. coli was grown in Luria-Bertani (LB) medium (30).Construction of plasmids. Construction of pTf85. A member of family 1 repeated DNA from A. ferrooxidans ATCC 19859 was cloned into pBR322, and the resulting plasmid was designated pTf11 (50). An internal SphI fragment of pTf1 1 was subcloned into pBR322 and termed pTf1 1-sph. pTf1 1-sph was subsequently used as a probe in a Southern blot...
Fe-S clusters are versatile and essential cofactors that participate in multiple and fundamental biological processes. In Escherichia coli , the biogenesis of these cofactors requires either the housekeeping Isc pathway, or the stress-induced Suf pathway which plays a general role under conditions of oxidative stress or iron limitation. In the present work, the Fe-S cluster assembly Isc and Suf systems of acidophilic Bacteria and Archaea, which thrive in highly oxidative environments, were studied. This analysis revealed that acidophilic microorganisms have a complete set of genes encoding for a single system (either Suf or Isc). In acidophilic Proteobacteria and Nitrospirae, a complete set of isc genes ( iscRSUAX-hscBA-fdx ), but not genes coding for the Suf system, was detected. The activity of the Isc system was studied in Leptospirillum sp. CF-1 (Nitrospirae). RT-PCR experiments showed that eight candidate genes were co-transcribed and conform the isc operon in this strain. Additionally, RT-qPCR assays showed that the expression of the iscS gene was significantly up-regulated in cells exposed to oxidative stress imposed by 260 mM Fe 2 (SO 4 ) 3 for 1 h or iron starvation for 3 h. The activity of cysteine desulfurase (IscS) in CF-1 cell extracts was also up-regulated under such conditions. Thus, the Isc system from Leptospirillum sp. CF-1 seems to play an active role in stressful environments. These results contribute to a better understanding of the distribution and role of Fe-S cluster protein biogenesis systems in organisms that thrive in extreme environmental conditions.
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