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The terrestric oligochaete worm Enchytraeus buchholzi survives in cadmium (Cd)-polluted environments by aid of its Cd-inducible 25 kDa cysteine-rich protein (CRP). Here, we analyze promoter and structure of the crp gene and compare its relationship to MT genes. The crp gene, approximately 12 kbp long, consists of 10 exons with exons 2 to 9 encoding eight almost identical repeats of predominantly 31 amino acids of the CRP. The introns of the crp gene contain various repetitive elements including retrotransposon-like sequences. The 683-bp promoter of the non-constitutive crp gene exhibits a much higher basal activity than the mouse MT-II promoter in HepG2 cells. Essential for crp promoter activity is the distal region (-683/-521) with a GC box and the proximal region (-308/-8) with the four MREa, b, c, d and AP-1, -2, -3 elements, whereas the central portion (-521/-309) with CAAT box, CRE and a XRE causes promoter repression. The TATA box-, MREc- and the AP-2, -3-containing region are required for high crp promoter activity. Our data support the view that the crp gene is a unique MT-gene and has evolved by exon duplications from a MT-like ancestral gene.

The ubiquitous earthworm Enchytraeus buchholzi is provided with a very effective, but still unknown, mechanism of Cadmium (Cd)-detoxification, a central role in which has been ascribed to the Cd-inducible crp-gene encoding a novel cysteine-rich, non-metallothionein 25 kDa protein. This study identifies another Cd-responsive gene by differential screening of a cDNA-library constructed from Cd-exposed E. buchholzi. The isolated cDNA-clone designated Ebaldh encodes a putative aldehyde dehydrogenase. Northern blot analysis shows that the Ebaldh-expression is strongly enhanced by Cd, but remains unaffected by other stressors such as Zn, Hg, and H2O2. This Cd-specificity of Ebaldh suggests that Cd-detoxification in E. buchholzi requires low intracellular concentrations of aldehydes which are known to target sulfhydryl groups thus inactivating the Cd-binding capacity of CRP.

The expression of a novel Cd-inducible gene encoding a 25 kDa non-metallothionein cysteine-rich protein (CRP) in the earthworm Enchytraeus buchholzi is investigated with respect to Cd-specificity and Cd-sensitivity. The amount of CRP-mRNA correlates with the environmental as well as with the intra-worm Cd-concentration. Even the subtoxic concentration of 100 microgram Cd/1 induces CRP-gene expression. CRP-mRNA is expressed already 2 h after the worms were exposed to Cd. Other heavy metals or stress conditions can induce only little or even no CRP-mRNA. The CRP-gene is switched on long before Cd becomes toxic to worms and can be therefore taken as a warning signal of prospective irreversible Cd-damages at an early, still reversible stage.

Cadmium accumulation and its effect on gene expression have been investigated at sublethal cadmium concentrations in the soil oligochaete Enchytraeus buchholzi. This worm is capable of accumulating cadmium to large amounts, which coincides with the induction of a mRNA isolated as a cDNA clone by differential screening of a cDNA library constructed from cadmium-treated enchytraeids. The cDNA clone designated CRP1 is 1474 base pairs in length and contains a 753-base pair open reading frame, encoding a novel Cys-rich non-metallothionein protein. In vitro translation of the in vitro transcribed CRP1 results in a protein with a molecular mass of 25 kDa and an pI of approximately 7.5. These values are consistent with those predicted from the deduced amino acid sequence. The CRP protein contains 27% Cys, most of them arranged in Cys-X-Cys and Cys-Cys segments. The sequence is also characterized by a 31-amino-acid motif, which is tandemly repeated along the sequence. Northern blot analysis reveals that the CRP gene is not constitutively expressed in untreated worms, but rather it is rapidly induced by cadmium. The CRP gene may be a promising candidate gene for monitoring bioavailable cadmium at subtoxic levels in terrestric environments.

Enchytraeus buchholzi (Oligochaeta) were exposed to various concentrations of CdCl2 in agar and aqueous solution. The Cd uptake was determined by atomic absorption spectroscopy as well as Cd effects on survival, reproduction, and mRNA synthesis by in vitro translation of total RNA in a rabbit reticulocyte lysate system. Although Cd was rapidly accumulated by the worms, any acute toxic Cd effects at concentrations below 4 mg Cd/liter were not detectable. However, such subtoxic Cd concentrations caused the induction of an mRNA species coding for a nonmetallothionein 33-kDa protein as revealed by 2D electrophoresis of in vitro translated in vitro translated proteins using [35S]methionine. The Cd-induced synthesis of this transcriptionally regulated protein might be a preindicator for a Cd intoxication in enchytraeids.