Characteristics of the metatranscriptomic libraries used for comparative diversity analysis.

<p>Average values from replicates are shown.</p

Linkage of community diversity to metabolic function.

<p>(a) Relative abundance of mRNA reads matching selected genomes from bacteria with relevant characteristic metabolisms: iron oxidation (red), methanotrophy (blue) and anaerobic iron respiration (green). (b) Relative abundance of <i>pmo</i>CAB matching transcripts coding for the three subunits of the methane monooxygenase. Error bars correspond to standard deviation from the replicates.</p

Relative abundance and distribution of microbial taxa in the SSU and LSU rRNA transcript libraries.

<p>The rRNA reads from each triplicate are combined and average values shown. (a) Comparison of the five samples at the phylum level. (b) Comparison of the combined two surfaces (S1 and S2) and the subjacent depth (D1 and D2) samples at the phylum, class and order level. Error bars indicate the standard deviations among the replicates.</p

Schematic profile of the iron rich microbial mat with sampling sites.

<p>Schematic profile of the iron rich microbial mat with sampling sites.</p

A schematic representation of the Hap4 and Yap1 proteins.

<p>The <i>Saccharomyces cerevisiae</i> Hap4 and Yap1 proteins are schematically represented relative to the two <i>Hansenula polymorpha</i> HpHap4-A and HpHap4-B ones. The main motifs are indicated in grey (N-terminal Hap4), black (bZIP of BR motif; the BR motif is the DNA binding part of the bZIP motif) and striped (CRD or cysteine rich domain).</p

Identification of a “Relic sequence” in <i>S. cerevisiae</i>.

<p>Upper part (Hap4) is the N-ter Hap4 motif with small extensions (the motif itself spans aminoacids 60 to 76). Bottom part (Yap7) is a small sequence in the Yap7 protein which is 68% similar to the upper sequence. Yap7 is a member of the bZIP family of <i>S. cerevisiae</i> transactivators. Its function is not precisely known <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112263#pone.0112263-RodriguesPousada1" target="_blank">[50]</a>. Refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112263#pone-0112263-g001" target="_blank">Fig 1</a> for the various motifs of these proteins.</p

qRT-PCR of <i>HpHAP4-A</i> and <i>HpHAP4-B</i> regulation of gene expression in <i>H. polymorpha.</i>

<p>The P(H1) value indicates the probability that the difference between the sample and control groups is due only by chance and was analysed with the REST software (Qiagen, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112263#s2" target="_blank">Material and Methods</a> for more details). Statistically significant results are shown in bold. The case of AAD2* is not conclusive since the data were obtained in two different experiments (two qRT-PCRs, regulated and reproducible and a third one, performed later, which was not).</p><p>qRT-PCR of <i>HpHAP4-A</i> and <i>HpHAP4-B</i> regulation of gene expression in <i>H. polymorpha.</i></p

List of <i>H. polymorpha</i> genes and corresponding primers used for qRT-PCR.

<p>* The <i>H. polymorpha</i> gene names are given according to their homologues in <i>S. cerevisiae.</i></p><p>**In the case of the Aryl-alcohol dehydrogenase genes (<i>AAD</i> genes, seven in Sc) we found only two genes in Hp and tested both. Priming with the AAD1 oligomers did not work and the experiment was not carried further.</p><p>List of <i>H. polymorpha</i> genes and corresponding primers used for qRT-PCR.</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

Functional Study of the Hap4-Like Genes Suggests That the Key Regulators of Carbon Metabolism <i>HAP4</i> and Oxidative Stress Response <i>YAP1</i> in Yeast Diverged from a Common Ancestor

<div><p>The transcriptional regulator <i>HAP4</i>, induced by respiratory substrates, is involved in the balance between fermentation and respiration in <i>S. cerevisiae</i>. We identified putative orthologues of the Hap4 protein in all ascomycetes, based only on a conserved sixteen amino acid-long motif. In addition to this motif, some of these proteins contain a DNA-binding motif of the bZIP type, while being nonetheless globally highly divergent. The genome of the yeast <i>Hansenula polymorpha</i> contains two <i>HAP4</i>-like genes encoding the protein HpHap4-A which, like ScHap4, is devoid of a bZIP motif, and HpHap4-B which contains it. This species has been chosen for a detailed examination of their respective properties. Based mostly on global gene expression studies performed in the <i>S. cerevisiae HAP4</i> disruption mutant (<i>ScΔhap4</i>), we show here that HpHap4-A is functionally equivalent to ScHap4, whereas HpHap4-B is not. Moreover <i>HpHAP4-B</i> is able to complement the H₂O₂ hypersensitivity of the <i>ScYap1</i> deletant, <i>YAP1</i> being, in <i>S. cerevisiae</i>, the main regulator of oxidative stress. Finally, a transcriptomic analysis performed in the <i>ScΔyap1</i> strain overexpressing <i>HpHAP4-B</i> shows that HpHap4-B acts both on oxidative stress response and carbohydrate metabolism in a manner different from both ScYap1 and ScHap4. Deletion of these two genes in their natural host, <i>H. polymorpha</i>, confirms that <i>HpHAP4-A</i> participates in the control of the fermentation/respiration balance, while <i>HpHAP4-B</i> is involved in oxidative stress since its deletion leads to hypersensitivity to H₂O₂. These data, placed in an evolutionary context, raise new questions concerning the evolution of the <i>HAP4</i> transcriptional regulation function and suggest that Yap1 and Hap4 have diverged from a unique regulatory protein in the fungal ancestor.</p></div