SummaryNADPH oxidases (Nox) have been characterized as higher eukaryotic enzymes used deliberately to produce reactive oxygen species (ROS). The recent discovery of new functional members of the Nox family in plants and animals has led to the recognition of the increasing importance of ROS as signals involved in regulation of diverse cellular processes such as defence, growth and signalling. Here, we address the role of NADPH oxidase-generated ROS in the biology of the filamentous fungus Aspergillus nidulans . We characterize the noxA gene and show that it encodes a member of a novel NADPH oxidase subfamily ubiquitous in lower eukaryotes. Deletion of noxA specifically blocks differentiation of sexual fruit bodies (cleistothecia), without affecting hyphal growth or asexual development. Accordingly, the noxA gene is induced during sexual development, peaking at the time of cleistothecia differentiation and in parallel with the hülle cell-associated catalase peroxidase gene cpeA . This expression pattern is not dependent on transcription factors SteA and StuA, which are essential for cleistothecia formation. In contrast, noxA -dependent premature sexual development correlates with noxA derepression in D D D D sakA null mutants, connecting stress MAPK signalling to the regulated production of ROS. Using a nitroblue tetrazolium (NBT) assay to detect superoxide, we found that hülle cells and cleistothecia initials produce superoxide in a process inhibited by NADPH oxidase inhibitor DPI and markedly reduced in D D D D noxA mutants. Furthermore, using H 2 DCFDA, we detected that H 2 O 2 and possibly other ROS are generated in a NoxA-dependent fashion, mainly in the external walls from cleistothecia initials. The essential role of NoxA-generated ROS in A. nidulans sexual differentiation and the presence of one or two noxA homologues in all analysed filamentous fungi suggest that NADPH oxidase-generated ROS play important roles in fungal physiology and differentiation.

Chromium is a non-essential and well-known toxic metal for microorganisms and plants. The widespread industrial use of this heavy metal has caused it to be considered as a serious environmental pollutant. Chromium exists in nature as two main species, the trivalent form, Cr(III), which is relatively innocuous, and the hexavalent form, Cr(VI), considered a more toxic species. At the intracellular level, however, Cr(III) seems to be responsible for most toxic effects of chromium. Cr(VI) is usually present as the oxyanion chromate. Inhibition of sulfate membrane transport and oxidative damage to biomolecules are associated with the toxic effects of chromate in bacteria. Several bacterial mechanisms of resistance to chromate have been reported. The best characterized mechanisms comprise efflux of chromate ions from the cell cytoplasm and reduction of Cr(VI) to Cr(III). Chromate efflux by the ChrA transporter has been established in Pseudomonas aeruginosa and Cupriavidus metallidurans (formerly Alcaligenes eutrophus) and consists of an energy-dependent process driven by the membrane potential. The CHR protein family, which includes putative ChrA orthologs, currently contains about 135 sequences from all three domains of life. Chromate reduction is carried out by chromate reductases from diverse bacterial species generating Cr(III) that may be detoxified by other mechanisms. Most characterized enzymes belong to the widespread NAD(P)H-dependent flavoprotein family of reductases. Several examples of bacterial systems protecting from the oxidative stress caused by chromate have been described. Other mechanisms of bacterial resistance to chromate involve the expression of components of the machinery for repair of DNA damage, and systems related to the homeostasis of iron and sulfur.

Sulfur is an essential element for microorganisms and it can be obtained from varied compounds, sulfate being the preferred source. The first step for sulfate assimilation, sulfate uptake, has been studied in several bacterial species. This article reviews the properties of different bacterial (and archaeal) transporters for sulfate, molybdate, and related oxyanions. Sulfate uptake is carried out by sulfate permeases that belong to the SulT (CysPTWA), SulP, CysP/(PiT), and CysZ families. The oxyanions molybdate, tungstate, selenate and chromate are structurally related to sulfate. Molybdate is transported mainly by the high-affinity ModABC system and tungstate by the TupABC and WtpABC systems. CysPTWA, ModABC, TupABC, and WtpABC are homologous ATP-binding cassette (ABC)-type transporters with similar organization and properties. Uptake of selenate and chromate oxyanions occurs mainly through sulfate permeases.

A comprehensive, structural and functional, in silico analysis of the medium-chain dehydrogenase/reductase (MDR) superfamily, including 583 proteins, was carried out by use of extensive database mining and the BLASTP program in an iterative manner to identify all known members of the superfamily. Based on phylogenetic, sequence, and functional similarities, the protein members of the MDR superfamily were classified into three different taxonomic categories: (a) subfamilies, consisting of a closed group containing a set of ideally orthologous proteins that perform the same function; (b) families, each comprising a cluster of monophyletic subfamilies that possess significant sequence identity among them and might share or not common substrates or mechanisms of reaction; and (c) macrofamilies, each comprising a cluster of monophyletic protein families with protein members from the three domains of life, which includes at least one subfamily member that displays activity related to a very ancient metabolic pathway. In this context, a superfamily is a group of homologous protein families (and/or macrofamilies) with monophyletic origin that shares at least a barely detectable sequence similarity, but showing the same 3D fold.The MDR superfamily encloses three macrofamilies, with eight families and 49 subfamilies. These subfamilies exhibit great functional diversity including noncatalytic members with different subcellular, phylogenetic, and species distributions. This results from constant enzymogenesis and proteinogenesis within each kingdom, and highlights the huge plasticity that MDR superfamily members possess. Thus, through evolution a great number of taxa-specific new functions were acquired by MDRs. The generation of new functions fulfilled by proteins, can be considered as the essence of protein evolution. The mechanisms of protein evolution inside MDR are not constrained to conserve substrate specificity and/or chemistry of catalysis. In consequence, MDR functional diversity is more complex than sequence diversity.MDR is a very ancient protein superfamily that existed in the last universal common ancestor. It had at least two (and probably three) different ancestral activities related to formaldehyde metabolism and alcoholic fermentation. Eukaryotic members of this superfamily are more related to bacterial than to archaeal members; horizontal gene transfer among the domains of life appears to be a rare event in modern organisms.Keywords: protein taxonomy; protein evolution; mediumchain alcohol dehydrogenase; enoyl reductase; formaldehyde dehydrogenase.Correspondence to H. Riveros-Rosas, Depto. Bioquı´mica, Fac. Medicina, UNAM, Apdo. Postal 70-159, Cd. Universitaria, Me´xico, 04510, D.F., Me´xico. Fax: + 52 55 5616 2419, Tel.: + 52 55 5622 0829, E-mail: hriveros@servidor.unam.mx Abbreviations: AADH, allyl alcohol dehydrogenase; ACR, acyl-CoA reductase; ADH, alcohol dehydrogenase; AL, alginate lyase; ARP, auxinregulated protein; AST, membrane traffic protein; BCHC, 2-desacetyl-2-hydroxyethyl bacteriochlorophyll...

Because toxic heavy metals (including chromium) have been abundant on the Earth since the beginning of life, microbes have been exposed to them for nearly four billion years [1]. In response, cells have developed diverse mechanisms that confer heavy metal resistance, such as, for example, the active extrusion of toxic metal cations [2,3]. This constitutes one of the beststudied bacterial mechanisms of resistance to chromium, which is based on the active efflux of chromate driven by the membrane potential [4,5] ChrA is a membrane protein that confers resistance to the toxic ion chromate through the energy-dependent chromate efflux from the cytoplasm. In the protein databases, ChrA is a member of the chromate ion transporter (CHR) superfamily, composed of at least several dozens of members, distributed in the three domains of life. The aim of this work was to perform a phylogenetic analysis of the CHR superfamily. An exhaustive search for ChrA homologous proteins was carried out at the National Center for Biotechnology Information database. One hundred and thirty-five sequences were identified as members of the CHR superfamily [77 long-chain sequences, or bidomains (LCHR), and 58 short-chain sequences, or monodomains (SCHR)], organized mainly as tandem pairs of genes whose resultant proteins probably possess oppositely oriented membrane topology. LCHR sequences were split into amino and carboxyl domains, and the resultant domains were aligned with the SCHR proteins. A phylogenetic tree was reconstructed using four different methods, obtaining similar results. The domains were grouped into three clusters: the SCHR proteins cluster, the amino domain cluster of LCHR proteins and the carboxyl domain cluster of LCHR proteins. These results, as well as differences in the genomic context of CHR proteins, enabled the proteins to be sorted into two families (SCHR and LCHR), and 10 subfamilies. Evidence was found suggesting an ancient origin of LCHR proteins from the fusion of two SCHR protein-encoding genes; however, some secondary events of fusion and fission may have occurred later. The separate distribution of the LCHR and SCHR proteins, differences in the genomic context in both groups and the fact that chromate transport has been demonstrated only in LCHR proteins suggest that the CHR proteins comprise two or more paralogous groups in the CHR superfamily.Abbreviations CHR, chromate ion transporter; ME, minimum evolution; MP, maximum parsimony; NJ, neighbour joining; TMS, transmembrane segment; UPGMA, unweighted pair-group method using arithmetic averages.

Protein profiles of mitochondria isolated from the heterotrophic chlorophyte Polytomella sp. grown on ethanol at pH 6.0 and pH 3.7 were analyzed by Blue Native and denaturing polyacrylamide gel electrophoresis. Steady-state levels of oxidative phosphorylation complexes were influenced by external pH. Levels of an abundant, soluble, mitochondrial protein of 85 kDa and its corresponding mRNA increased at pH 6.0 relative to pH 3.7. N-terminal and internal sequencing of the 85 kDa mitochondrial protein together with the corresponding cDNA identified it as a bifunctional aldehyde/alcohol dehydrogenase (ADHE) with strong similarity to homologues from eubacteria and amitochondriate protists. A mitochondrial targeting sequence of 27 amino acids precedes the N-terminus of the mature mitochondrial protein. A gene encoding an ADHE homologue was also identified in the genome of Chlamydomonas reinhardtii, a photosynthetic relative of Polytomella. ADHE reveals a complex picture of sequence similarity among homologues. The lack of ADHE from archaebacteria indicates a eubacterial origin for the eukaryotic enzyme. Among eukaryotes, ADHE has hitherto been characteristic of anaerobes since it is essential to cytosolic energy metabolism of amitochondriate protists such as Giardia intestinalis and Entamoeba histolytica. Its abundance and expression pattern suggest an important role for ADHE in mitochondrial metabolism of Polytomella under the conditions studied. The current data are compatible with the view that Polytomella ADHE could be involved either in ethanol production or assimilation, or both, depending upon environmental conditions. Presence of ADHE in an oxygen-respiring algal mitochondrion and co-expression at ambient oxygen levels with respiratory chain components is unexpected with respect to the view that eukaryotes acquired ADHE genes specifically as an adaptation to an anaerobic lifestyle.

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