Iron is an essential nutrient for sustaining bacterial growth; however, little is known about the molecular mechanisms that govern gene expression during the homeostatic response to iron availability. In this study we analyzed the global transcriptional response of Enterococcus faecalis to a non-toxic iron excess in order to identify the set of genes that respond to an increment of intracellular iron. Our results showed an up-regulation of transcriptional regulators of the Fur family (PerR and ZurR), the cation efflux family (CzcD) and ferredoxin, while proton-dependent Mn/Fe (MntH) transporters and the universal stress protein (UspA) were down-regulated. This indicated that E. faecalis was able to activate a transcriptional response while growing in the presence of an excess of non-toxic iron, assuring the maintenance of iron homeostasis. Gene expression analysis of E. faecalis treated with H(2)O(2) indicated that a fraction of the transcriptional changes induced by iron appears to be mediated by oxidative stress. A comparison of our transcriptomic data with a recently reported set of differentially expressed genes in E. faecalis grown in blood, revealed an important fraction of common genes. In particular, genes associated to oxidative stress were up-regulated in both conditions, while genes encoding the iron uptake system (feo and ycl operons) were up-regulated when cells were grown in blood. This suggested that blood cultures mimic an iron deficit, and was corroborated by measuring feo and ycl expression in E. faecalis treated with the iron chelating agent 2,2-dipyridil. In summary, our group identified an adaptive transcriptional mechanism in response to metal ion stress in E. faecalis, providing a foundation for future in-depth functional studies of the iron-activated regulatory network.

In this work we investigated the adaptive response of E. faecalis to Cu and the role of CopY, a Cu-dependent repressor, in the regulation of Cu metabolism. In doing so, we examined the whole-genome transcriptional response of E. faecalis wild-type (WT) and a ΔcopY strain exposed to non-toxic Cu excess. The results indicated that after Cu exposure, most of the genes that displayed a significant change in their expression levels in the WT strain (135 of the 145 up-regulated genes and 115 of the 142 down-regulated genes) were also differentially expressed in the E. faecalis ΔcopY strain. This extensive overlap in the transcriptional response, suggested that additional transcription factors mediate the response of E. faecalis to Cu. As a first step to analyze this possibility, we selected among the up-regulated genes five genes encoding putative transcriptional regulators and determined their expression levels at different times after Cu exposure. The temporal expression of these regulators was different from that of copY, which reached its maximum at the earliest time measured. Nevertheless, transcription elongation factor GreA, and members of Rrf2, Cro/CI and SorC/DeoR transcription factor families were induced shortly after Cu exposure, suggesting that these proteins are able to complement the role of CopY in the regulatory network activated by Cu. To our knowledge, this is the first report on the global transcriptional response to Cu in a member of this taxonomic group.

Classic copper indicators are not sensitive and specific for detecting excess copper exposure when this is higher than customary but not markedly elevated. Serum copper and ceruloplasmin (Cp) are the most commonly used indicators to assess nutritional status of copper. The objective of this paper was to study the influence of estrogens on these indicators and others used to assess early effects of excess copper exposure in humans and the expression of a set of copper related proteins in a hepatic cellular model. For the studies in humans, 107 healthy participants (18-50 years) were allocated as follows: group 1 (n = 39), women assessed on day 7 of their hormonal cycle; group 2 (n = 34), women assessed on day 21 of their hormonal cycle, and group 3 (n = 34, comparison group), healthy men. Participants received 8 mg Cu/day (as copper sulfate) during 6 months. Serum Cp and Cu, Cu-Zn-superoxide dismutase activity, liver function indicators [aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma glutamyltransferase (GGT)], and serum Fe and Zn concentrations were measured monthly. In addition, the influence of estradiol on intracellular total copper content, hctr1, dmt1 and shbg mRNA abundance and hCTR1, and DMT1 expression was measured in HepG2 cells. Serum Cu, Fe, and Zn and liver aminotransferases but not Cu-Zn-superoxide dismutase varied depending on sex. Fe nutrition indicators, GGT, and ALT activities showed significant differences between the hormonal phases. Cellular experiments showed that estradiol increased cellular Cu concentration and hCTR1 and DMT1 mRNA expression and changed these proteins expression patterns. Estradiols significantly influence the responses to copper at the whole body and the cellular levels, suggesting that they help maintaining copper availability for metabolic needs.

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