The Neurospora crassa chr-1 gene is up-regulated by chromate and its encoded CHR-1 protein causes chromate sensitivity and chromium accumulation

Abstract

The ChrA membrane protein belongs to the CHR superfamily of chromate ion transporters, which includes homologues from bacteria, archaea and eukaryotes. Bacterial ChrA homologues confer chromate resistance by exporting chromate ions from the cell's cytoplasm. The Neurospora crassa strain 74-A chr-1 gene encodes a putative CHR-1 protein of 507 amino acid residues, which belongs to the CHR superfamily. RT-PCR assays showed that expression of the chr-1 gene was up-regulated by chromate exposure of N. crassa cultures. Introduction in N. crassa of sense and antisense fragments of the chr-1 gene, as part of a silencing module within the pSilent-1 vector, produced transformants with a phenotype of resistance to chromate and diminished accumulation of chromium, as compared with the control strain containing only the vector. A chromate-resistance phenotype was also observed in N crassa strains deleted in the genomic chr-1 gene, thus confirming that the absence of CHR-1 protein confers chromate resistance to the fungus. The cDNA from N. crassa chr-1 gene (Ncchr-1) was cloned into the pYES2 vector under the control of a GAL promoter and the resulting recombinant plasmid was transferred to the yeast Saccharomyces cerevisiae. Galactose-induced S. cerevisiae transformants expressing Ncchr-1 were more sensitive to chromate and accumulated 2.5 times more chromium than the induced strain containing only the vector. Excess sulfate, a chromate analog, was unable to protect S. cerevisiae chr-1 transformants from chromate toxicity. These data indicate that the N. crassa CHR-1 protein functions as a transporter that takes up chromate; it also appears that this transport occurs in a sulfate-independent fashion. This is the first report assigning a role as a chromate transporter to a nonbacterial CHR protein.

Molecular mechanisms of Cr(VI) resistance in bacteria and fungi

“…CHR-1 protein (homologous to ChrA) is present in several ascomycetes and other fungal phyla, such as Basidiomycota and Zygomycota, whereas CHR-1 is absent in the vast majority of yeasts (Flores-Alvarez et al, 2012). To our knowledge, Neurospora crassa is the only fungus in which a CHR-1 protein has been characterized (Flores-Alvarez et al, 2012). The expression of the CHR-1 gene was induced upon Cr(VI) exposure, but the encoded protein elicited the opposite effect as the expected.…”

“…The expression of the CHR-1 gene was induced upon Cr(VI) exposure, but the encoded protein elicited the opposite effect as the expected. Instead of chromate tolerance, CHR-1 expression resulted in an increase in Cr(VI) sensitivity, indicating that it is a chromate transporter but promotes chromate accumulation inside the cell instead of extrusion (Flores-Alvarez et al, 2012). A possibility for the role of chromate uptake for the Chr-1 protein in N. crassa may be that the transported chromate is then destined for vacuolar compartmentalization.…”

“…CRDs have been identified in Archaea , Bacteria and Eukarya (Nies et al ., ; Flores‐Alvarez et al ., ), and consist of genes belonging to the chromate ion transport (CHR) superfamily (Ramirez‐Diaz et al ., ). Generally, CRDs include the chrA gene, which encodes a putative chromate efflux protein driven by the membrane potential (Pimentel et al ., ).…”

The guanidine thiocyanate‐high EDTA method for total microbial RNA extraction from severely heavy metal‐contaminated soils

“…4C). This is consistent with previous studies that observed an increase in the expression levels of chrA and yieF after Cr(VI) treatment in pure bacteria or cells (Flores‐Alvarez et al ., 2012; Liu et al ., 2015). However, due to the severe degradation of RNA extracted by the original GTHE approach, the expressions of chrA and yieF were very low, and even showed downregulation after Cr(VI) treatment (Fig.…”

Do environmentally induced DNA variations mediate adaptation in Aspergillus flavus exposed to chromium stress in tannery sludge?

“…In the Neurospora crassa strain 74-A, chr-1 gene that encodes a putative CHR-1 protein and belongs to the CHR superfamily was identified [ 33 ]. However, contrary to the bacterial ChrA (chromate transport protein) homologues that confer chromate resistance by exporting chromate ions from the cell’s cytoplasm, the experimental data suggested that the N. crassa CHR-1 protein functions as a transporter that takes up chromate [ 34 ]. The presence of CHR-1 protein was reported to cause chromate sensitivity and chromium accumulation in N. crassa .…”