A subset of the diverse COG0523 family of putative metal chaperones is linked to zinc homeostasis in all kingdoms of life

<p>Abstract</p> <p>Background</p> <p>COG0523 proteins are, like the nickel chaperones of the UreG family, part of the G3E family of GTPases linking them to metallocenter biosynthesis. Even though the first COG0523-encoding gene, <it>cobW</it>, was identified almost 20 years ago, little is known concerning the function of other members belonging to this ubiquitous family.</p> <p>Results</p> <p>Based on a combination of comparative genomics, literature and phylogenetic analyses and experimental validations, the COG0523 family can be separated into at least fifteen subgroups. The CobW subgroup involved in cobalamin synthesis represents only one small sub-fraction of the family. Another, larger subgroup, is suggested to play a predominant role in the response to zinc limitation based on the presence of the corresponding COG0523-encoding genes downstream from putative Zur binding sites in many bacterial genomes. Zur binding sites in these genomes are also associated with candidate zinc-independent paralogs of zinc-dependent enzymes. Finally, the potential role of COG0523 in zinc homeostasis is not limited to Bacteria. We have predicted a link between COG0523 and regulation by zinc in Archaea and show that two COG0523 genes are induced upon zinc depletion in a eukaryotic reference organism, <it>Chlamydomonas reinhardtii</it>.</p> <p>Conclusion</p> <p>This work lays the foundation for the pursuit by experimental methods of the specific role of COG0523 members in metal trafficking. Based on phylogeny and comparative genomics, both the metal specificity and the protein target(s) might vary from one COG0523 subgroup to another. Additionally, Zur-dependent expression of <it>COG0523 </it>and putative paralogs of zinc-dependent proteins may represent a mechanism for hierarchal zinc distribution and zinc sparing in the face of inadequate zinc nutrition.</p

Copper Response Element and Crr1-Dependent Ni(2+)-Responsive Promoter for Induced, Reversible Gene Expression in Chlamydomonas reinhardtii

The Cpx1 and Cyc6 genes of Chlamydomonas reinhardtii are activated in copper-deficient cells via a signal transduction pathway that requires copper response elements (CuREs) and a copper response regulator defined by the CRR1 locus. The two genes can also be activated by provision of nickel or cobalt ions in the medium. The response to nickel ions requires at least one CuRE and also CRR1 function, suggesting that nickel interferes with a component in the nutritional copper signal transduction pathway. Nickel does not act by preventing copper uptake/utilization because (i) holoplastocyanin formation is unaffected in Ni(2+)-treated cells and (ii) provision of excess copper cannot reverse the Ni-dependent activation of the target genes. The CuRE is sufficient for conferring Ni-responsive expression to a reporter gene, which suggests that the system has practical application as a vehicle for inducible gene expression. The inducer can be removed either by replacing the medium or by chelating the inducer with excess EDTA, either of which treatments reverses the activation of the target genes

FEA1, FEA2, and FRE1, Encoding Two Homologous Secreted Proteins and a Candidate Ferrireductase, Are Expressed Coordinately with FOX1 and FTR1 in Iron-Deficient Chlamydomonas reinhardtii▿

Previously, we had identified FOX1 and FTR1 as iron deficiency-inducible components of a high-affinity copper-dependent iron uptake pathway in Chlamydomonas. In this work, we survey the version 3.0 draft genome to identify a ferrireductase, FRE1, and two ZIP family proteins, IRT1 and IRT2, as candidate ferrous transporters based on their increased expression in iron-deficient versus iron-replete cells. In a parallel proteomic approach, we identified FEA1 and FEA2 as the major proteins secreted by iron-deficient Chlamydomonas reinhardtii. The recovery of FEA1 and FEA2 from the medium of Chlamydomonas strain CC425 cultures is strictly correlated with iron nutrition status, and the accumulation of the corresponding mRNAs parallels that of the Chlamydomonas FOX1 and FTR1 mRNAs, although the magnitude of regulation is more dramatic for the FEA genes. Like the FOX1 and FTR1 genes, the FEA genes do not respond to copper, zinc, or manganese deficiency. The 5′ flanking untranscribed sequences from the FEA1, FTR1, and FOX1 genes confer iron deficiency-dependent expression of ARS2, suggesting that the iron assimilation pathway is under transcriptional control by iron nutrition. Genetic analysis suggests that the secreted proteins FEA1 and FEA2 facilitate high-affinity iron uptake, perhaps by concentrating iron in the vicinity of the cell. Homologues of FEA1 and FRE1 were identified previously as high-CO2-responsive genes, HCR1 and HCR2, in Chlorococcum littorale, suggesting that components of the iron assimilation pathway are responsive to carbon nutrition. These iron response components are placed in a proposed iron assimilation pathway for Chlamydomonas

Ni induces the CRR1-dependent regulon revealing overlap and distinction between hypoxia and Cu deficiency responses in Chlamydomonas reinhardtii

The selectivity of metal sensors for a single metal ion is critical for cellular metal homeostasis. A suite of metal-responsive regulators is required to maintain a prescribed balance of metal ions ensuring that each apo-protein binds the correct metal. However, there are cases when non-essential metals ions disrupt proper metal sensing. An analysis of the Ni-responsive transcriptome of the green alga Chlamydomonas reinhardtii reveals that Ni artificially turns on the CRR1-dependent Cu-response regulon. Since this regulon also responds to hypoxia, a combinatorial transcriptome analysis was leveraged to gain insight into the mechanisms by which Ni interferes with the homeostatic regulation of Cu and oxygen status. Based on parallels with the effect of Ni on the hypoxic response in animals, we propose that a possible link between Cu, oxygen and Ni sensing is an as yet uncharacterized prolyl hydroxylase that regulates a co-activator of CRR1. This analysis also identified transcriptional responses to the pharmacological activation of the Cu-deficiency regulon. Although the Ni-responsive CRR1 regulon is composed of 56 genes (defined as the primary response), 259 transcripts responded to Ni treatment only when a copy of the wild-type CRR1 gene was present. The genome-wide impact of CRR1 target genes on the transcriptome was also evident from the 210 transcripts that were at least 2-fold higher in the crr1 strain, where the abundance of many CRR1 targets was suppressed. Additionally, we identified 120 transcripts that responded to Ni independent of CRR1 function. The putative functions of the proteins encoded by these transcripts suggest that high Ni results in protein damage