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• Cross-talk between different Yap factors

• Nitrosative stress response in Desulfovibrio gigas

• Desulfovibrio gigas genome: past and future

Metals are of the most important environmental toxics that cause acute and chronic adverse health effects including cancer. In the last years we have been focused on the mechanisms used by eukaryotic cells to overcome metal toxicity. We are particularly interested in the general aspects of stress response in Saccharomyces cerevisiae orchestrated by different transcription factors that trigger the reprogramming of gene expression in response either to toxic metal or to imbalance homeostasis of essential metals.

Metals are of the most important environmental toxics that cause acute and chronic adverse health effects including cancer; among these arsenic compounds are considered the greatest single-cause of ill-health in the world. Indeed, prenatal arsenic exposure has been found to cause genome-wide changes in human newborns.The ubiquity of arsenic in the environment allowed the evolution of very similar defense mechanisms in organisms ranging from bacteria to man. Indeed, from 20 specific As(V)-sensitive mutants in mitochondrial genes found, 13 genes have orthologs in humans. Furthermore, the genome-wide set of S. cerevisiae deletion strains provided the opportunity to understand the mechanisms by which arsenic trioxide selectively kills acute promyelocytic leukemia cells.

Arsenic (As) compounds are considered the greatest single-cause of ill-health in the world. Indeed, prenatal arsenic exposure has been found to cause genome-wide changes in human newborns. The ubiquity of arsenic in the environment allowed the evolution of very similar defense mechanisms in organisms ranging from bacteria to man. Thirteen out of the twenty yeast mitochondrial genes previously involved in As (V) toxicity were shown to have orthologs in humans. As such S. cerevisiae deletion strains are powerful tools to understand the mechanisms by which arsenic trioxide selectively kills acute promyelocytic leukemia cells. Within this frame, we have recently shown that Arsenic compounds disturb iron homeostasis in yeast as well as in mammalian cells (figure above). This finding can be relevant to future clinical applications. Yap8, one of the eight members of the Yap family of transcription factors, is a key regulator in yeast of the expression of two genes encoding the arsenate-reductase Acr2 and the plasma membrane arsenite efflux transporter Acr3. These two genes form the Yap8 regulon and we have recently addressed the study of its very restricted DNA binding specificity using a model of Yap8-DNA interaction and in vivo and in vitro experimental approaches. Acr2 and Acr3 constitute the main Arsenics detoxification pathway. Yap1, the master regulator of the cell antioxidant defenses, contributes to arsenic stress responses by regulating the expression of a vacuolar arsenite detoxification pathway encoded by YCF1, although we have recently shown that the most important role of Yap1 in arsenic adaptation is through the maintenance of the redox homeostasis disturbed by inorganic arsenic compounds. To analyze whether Yap1 and Yap8 use similar mechanisms to transduce the stress signals to the basal transcription machinery, we are addressing the effect of mutations in specific subunits of the tail module of the mediator complex.
Yap1 appears to be in the forefront of cadmium stress response. Yap2 is the family member that shares the highest homology with Yap1, but very little is known about its functional role. Nevertheless it is known that, when overexpressed, Yap2 confers resistance to several stress agents, such as cadmium. Given Yap1 and Yap2 similarities it is widely accepted that a certain degree of functional overlap exists between both transcription factors, however it is evident that Yap2 plays unique roles without interference of the realm of Yap1 function. Current investigations in our laboratory are aimed at understanding of how Yap1 and Yap2 coordinate their gene target expression, in order to endow the cell with the ability to overcome cadmium toxicity.

Cobalt has a rare occurrence in nature, but may accumulate in cells to toxic levels. We have shown that Cobalt activates Yap1 that alleviate the oxidative damage caused by this metal. Yap1 partially regulates cobalt cellular uptake via the regulation of the high affinity phosphate transporter Pho84. Transcriptomic analysis revealed that Yap1 is a repressor of the low affinity iron transport Fet4. Although Fet4 repression by Yap1 has no effect on cobalt uptake we are currently investigating whether it may be its first line of defense against other toxic metals.

Iron (Fe) is an essential metal to most forms of life. However, the same chemical properties that make Fe such a central element for life also make it a strong pro-oxidant that can generate powerful reactive oxygen species (ROS) through Fenton type reactions. Unlike humans, but similar to plants, the yeast cell vacuoles function as iron reservoirs. In yeast, iron storage is mediated by Ccc1, a vacuolar transporter that effects the accumulation of iron in the vacuoles. In a high-Fe milieu, CCC1 gene deletion is lethal and its expression is regulated by the transcription factor Yap5. We have analysed the transcriptional response of the yap5 null mutant subjected to high concentrations of iron, having identified GRX4 gene as a Yap5 target. This gene encodes a monothiol glutaredoxin previously known to be involved in the regulation Aft1 nuclear localization. Consistently we have shown that in the absence of Yap5, Aft1 nuclear exclusion is slightly impaired.

Overall our recent studies provide further evidence that cells control metal homeostasis or metal toxicity by using multiple pathways.

Although the binding of promoters by multiple transcriptional regulators has been characterized as a specific feature of higher eukaryotes, many yeast genes were found in simultaneous association with several transcription factors. Within this context, Yap4 has been shown to be regulated either by Msn2 under conditions of osmotic stress or by Msn2 and Yap1 under oxidative stress. Using computational approaches, transcriptional regulatory networks were derived that were shown to control several cellular processes such as the cell cycle, environmental stress, development and metabolism.

It was also predicted through mathematic modeling that Yap4 interacts with Yap6. Several studies have shown that the Yap family members appear to have a degree of functional overlap as well as distinct physiological roles. It was indeed shown that it is possible, using DNA microarrays, to distinguish between the functions of Yap1 and Yap2. These two transcription factors although having overlapping functions, both activate non-overlapping gene sets.

One possible model put forward to describe the Yap network is that during oxidative stress the Yap1 and Yap2 homodimers activate distinct regulons whereas Yap1/Yap2 heterodimers collaborate to repress a separate regulon. Similarly, the yap8yap1 strain has increased metalloid sensitivity than either single mutant. The expression of the Yap8 target genes ACR2, ACR3 and YCF1 is also significantly regulated by Yap1, both of which have been shown to bind the YRE variant, TGATTAATAATCA. We have constructed a Yap0 mutant strain, a strain deleted in all the Yap factors which is a valuable tool to discriminate overlapping functions as well as the interplay between them.

Desulfovibrio gigas is an anaerobic microorganism that belongs to the group of sulfate reducing bacteria (SRB). SRB are metabolic versatile microorganisms widespread in nature as well as in the human gastrointestinal tract, being often exposed to reactive nitrogen species (RNS), produced by other bacteria or by the human innate immune system. To cope with RNS deleterious effects microorganisms have developed several mechanisms that afford protection against nitrosative stress. Although Desulfovibrio is the most studied genus of SRB, the mechanisms and regulatory elements involved in this protection are still poorly understood. Using D. gigas as a model organism we addressed the importance of several transcriptional factors in nitrosative stress response. Our lab was pioneer in constructing mutants of this bacterium, and we are using this approach to investigate whether those factors are involved in NO detoxification.

In Desulfovibrio gigas we have already shown that the Rubredoxin oxygen oxidoreductase, ROO, affords protection against nitrosative stress. Our recent findings have shown that ROO gene expression is regulated by NorR1L, an ortholog of NorR from E.coli. Furthermore we found a NorR1L paralog (NorR2L) within the genome of D. gigas whose function under stress is currently being investigated.

Lately, we have found that D.gigas HcpR, a transcription factor belonging to the family of CRP/FNR, regulates several genes encoding proteins involved in nitrite and nitrate metabolism. Accordingly, an hcpR mutant strain displays a growth sensitivity phenotype to NO, strongly supporting a relevant role of this factor under nitrosative stress. Moreover, we found that several Desulfovibrio spp. possess HcpR paralogs bringing to light the possibility that these species may exhibit higher tolerance to nitrosative stress. Detailed structural and functional analyses of the sequences need, however, to be evaluated in order to fully understand their role in stress response. Further work is in progress in order to clarify the role of HcpR paralog in D.gigas.

Within the context of dissecting the mechanisms of nitrosative stress adaptation in SRBs, we aim at determining the transcription profile of D.gigas Hcpr, NorR and their respective paralog genes null mutants, after challenge with NO.

Together with STAB VIDA Lda., we have recently sequenced the D. gigas genome providing insights into the integrated network of energy conserving complexes and structures present in this bacterium.

The genome of D. gigas (CP006585) consists of one circular chromosome of 3,693,899 base-pairs (bp) having 3,370 genes of which 3,273 are protein coding classified according to its predicted COG function. The genome has a G+C content of 63.4% that reflects a biased codon usage as such D. gigas prefers high G+C codons (66.87%), with a clear preference for cytosine (C) in the 3rd position. The genome is very compact as observed by its gene density of 1,128 bp per gene and the average length of each gene is 993 bp. Its genome contains solely 17 genes encoding transposases and only a single rRNA operon indicating a decreased genome rearrangement, as multiple rRNA operons serves as sites for homologous recombination. The plasmid of this bacterium (CP006586) has a size of 101,949bp, containing 75 ORFs, of which 72 are coding regions.

The size of Desulfovibrio gigas is larger than the one of other Desulfovibrio spp. Its length is of 5 to 10 µm and the width of 1.2 to 1.5 µm whereas the other species have a cell size of 3 to 5 µm by 0.5 to 1 µm. We have found several genes which can explain the large size of this bacterium

A survey of the genome of D. gigas for CRISPR repeats, revealed the presence of 6 CRISPR repeats with two of them being flanked by Cas operons. The CRISPRs are loci encompassing several short repeats functioning as an adaptive microbial immune system, that have also been shown to limit horizontal gene transfer by preventing conjugation and plasmid transformation.

Gene duplications were found such as those encoding fumarate reductase, formate dehydrogenase and superoxide dismutase. Complexes not yet described within Desulfovibrio genus were identified: Mnh complex, a v-type ATP-synthase as well as genes encoding the MinCDE system that could be responsible for the larger size of D. gigas when compared to other members of the genus. A low number of hydrogenases and the absence of the codh/acs and pfl genes, both present in D. vulgaris strains, indicate that intermediate cycling mechanisms may contribute substantially less to the energy gain in D. gigas compared to other Desulfovibrio spp. This might be compensated by the presence of other unique genomic arrangements of complexes such as the Rnf and the Hdr/Flox, or by the presence of NAD(P)H related complexes, like the Nuo, NfnAB or Mnh.

Structural representation of the circular chromosome (A) and plasmid (B) of Desulfovibrio gigas. Circular representations, from inside to the outside represent: (i) GC skew, richness of guanine over cytosine in the positive strand represented in green and cytosine over guanine represented in red; (ii) GC content, below average in purple, above average in gold; (iii) positive strand coding regions (below) and negative strand coding regions (above) colored according to COG functional terms of the best hit obtained from Blastp program; (iv) nucleotide position indicated in circular scale.

Taking advantage of the complete genome sequence is our future goal to have a general picture of the transcriptome of Desulfovibrio gigas subjected to different stress conditions that may interfere with its metabolism