Arsenate reduction and expression of multiple chromosomal ars operons in Geobacillus kaustophilus A1

Abstract

Geobacillus kaustophilus strain A1 was previously isolated from a geothermal environment for its ability to grow in the presence of high arsenate levels. In this study, the molecular mechanisms of arsenate resistance of the strain were investigated. As(V) was reduced to As(III), as shown by HPLC analysis. Consistent with the observation that the micro-organism is not capable of anaerobic growth, no respiratory arsenate reductases were identified. Using specific PCR primers based on the genome sequence of G. kaustophilus HTA426, three unlinked genes encoding detoxifying arsenate reductases were detected in strain A1. These genes were designated arsC1, arsC2 and arsC3. While arsC3 is a monocistronic locus, sequencing of the regions flanking arsC1 and arsC2 revealed the presence of additional genes encoding a putative arsenite transporter and an ArsR-like regulator upstream of each arsenate reductase, indicating the presence of sequences with putative roles in As(V) reduction, As(III) export and arsenic-responsive regulation. RT-PCR demonstrated that both sets of genes were co-transcribed. Furthermore, arsC1 and arsC2, monitored by quantitative real-time RT-PCR, were upregulated in response to As(V), while arsC3 was constitutively expressed at a low level. A mechanism for regulation of As(V) detoxification by Geobacillus that is both consistent with our findings and relevant to the biogeochemical cycle of arsenic and its mobility in the environment is proposed. INTRODUCTIONMicro-organisms have an important impact on the biogeochemical transformations of arsenic, and their activities affect the mobility and toxicity of this element. Elevated amounts of arsenic can occur, especially in geothermal environments (Stauffer & Thompson, 1984). Thus, thermophilic micro-organisms such as Geobacillus species that thrive in geothermal soils and sediments are of particular interest for studying the mechanisms for the detoxification of arsenic compounds.Genes for arsenic detoxification were first discovered and characterized in Gram-negative bacteria (Chen et al., 1986;Rosen, 1999). These genes are often plasmid-encoded and are widespread in prokaryotes (Bruhn et al., 1996;Ji & Silver, 1992b;Oden et al., 1994;Rosenstein et al., 1992). In thoroughly studied systems, the ars operon has been reported to contain the five genes arsRDABC, as in Escherichia coli (Cai & DuBow, 1996;Rosen et al., 1992), or at least the three genes arsRBC, as in Staphylococcus aureus (Silver et al., 1993). ArsA and ArsB are components of an arsenite-transporting ATPase, where ArsA is the ATPase and ArsB is the transmembrane component of the complex. In micro-organisms in which ArsA is absent, ArsB acts as a single-component transporter. Both ArsA and ArsB may confer resistance to arsenite and antimonite (Rosen, 1999). In its dimeric form, ArsR is an arsenite-responsive repressor that binds to the ars promoter (Xu & Rosen, 1997). ArsC is the arsenate reductase that converts arsenate to arsenite, conferring resistance to arsenate (Martin et al., 2001 Here w...

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From deep-sea volcanoes to human pathogens: a conserved quorum-sensing signal in Epsilonproteobacteria

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