Tracking the early events of photosymbiosis evolution.

Oxygenic photosynthesis evolved in cyanobacteria around 3.2 giga-annum (Ga) ago and was acquired by eukaryotes starting around 1.8 Ga ago by endosymbiosis. Photosymbiosis results either from integration of a photosynthetic bacteria by heterotrophic eukaryotes (primary photosymbiosis) or by successive integration of photosymbiotic eukaryotes by heterotrophic eukaryotes (secondary photosymbiosis). Primary endosymbiosis is thought to have been a rare event, whereas secondary and higher-order photosymbiosis evolved multiple times independently in different taxa. Despite its recurrent evolution, the molecular and cellular mechanisms underlying photosymbiosis are unknown. In this opinion, we discuss the primary events leading to the establishment of photosymbiosis, and we present recent research suggesting that, in some cases, domestication occurred instead of symbiosis, and how oxygen and host immunity can be involved in symbiont maintenance.

Mechanistic and functional aspects of the Ruminococcin C sactipeptide isoforms.

In a scenario where the discovery of new molecules to fight antibiotic resistance is a public health concern, ribosomally synthesized and post-translationally modified peptides constitute a promising alternative. In this context, the Gram-positive human gut symbiont Ruminococcus gnavus E1 produces five sactipeptides, Ruminococcins C1 to C5 (RumC1-C5), co-expressed with two radical SAM maturases. RumC1 has been shown to be effective against various multidrug resistant Gram-positives clinical isolates. Here, after adapting the biosynthesis protocol to obtain the four mature RumC2-5 we then evaluate their antibacterial activities. Establishing first that both maturases exhibit substrate tolerance, we then observed a variation in the antibacterial efficacy between the five isoforms. We established that all RumCs are safe for humans with interesting multifunctionalities. While no synergies where observed for the five RumCs, we found a synergistic action with conventional antibiotics targeting the cell wall. Finally, we identified crucial residues for antibacterial activity of RumC isoforms.

A Diverged Transcriptional Network for Usage of Two Fe-S Cluster Biogenesis Machineries in the Delta-Proteobacterium Myxococcus xanthus.

Myxococcus xanthus possesses two Fe-S cluster biogenesis machineries, ISC (iron-sulfur cluster) and SUF (sulfur mobilization). Here, we show that in comparison to the phylogenetically distant Enterobacteria, which also have both machineries, M. xanthus evolved an independent transcriptional scheme to coordinately regulate the expression of these machineries. This transcriptional response is directed by RisR, which we show to belong to a phylogenetically distant and biochemically distinct subgroup of the Rrf2 transcription factor family, in comparison to IscR that regulates the isc and suf operons in Enterobacteria. We report that RisR harbors an Fe-S cluster and that holo-RisR acts as a repressor of both the isc and suf operons, in contrast to Escherichia coli, where holo-IscR represses the isc operon whereas apo-IscR activates the suf operon. In addition, we establish that the nature of the cluster and the DNA binding sites of RisR, in the isc and suf operons, diverge from those of IscR. We further show that in M. xanthus, the two machineries appear to be fully interchangeable in maintaining housekeeping levels of Fe-S cluster biogenesis and in synthesizing the Fe-S cluster for their common regulator, RisR. We also demonstrate that in response to oxidative stress and iron limitation, transcriptional upregulation of the M. xanthus isc and suf operons was mediated solely by RisR and that the contribution of the SUF machinery was greater than the ISC machinery. Altogether, these findings shed light on the diversity of homeostatic mechanisms exploited by bacteria to coordinately use two Fe-S cluster biogenesis machineries. IMPORTANCE Fe-S proteins are ubiquitous and control a wide variety of key biological processes; therefore, maintaining Fe-S cluster homeostasis is an essential task for all organisms. Here, we provide the first example of how a bacterium from the Deltaproteobacteria branch coordinates expression of two Fe-S cluster biogenesis machineries. The results revealed a new model of coordination, highlighting the unique and common features that have independently emerged in phylogenetically distant bacteria to maintain Fe-S cluster homeostasis in response to environmental changes. Regulation is orchestrated by a previously uncharacterized transcriptional regulator, RisR, belonging to the Rrf2 superfamily, whose members are known to sense diverse environmental stresses frequently encountered by bacteria. Understanding how M. xanthus maintains Fe-S cluster homeostasis via RisR regulation revealed a strategy reflective of the aerobic lifestyle of this organsim. This new knowledge also paves the way to improve production of Fe-S-dependent secondary metabolites using M. xanthus as a chassis.

The noncoding RNA CcnA modulates the master cell cycle regulators CtrA and GcrA in Caulobacter crescentus.

Bacteria are powerful models for understanding how cells divide and accomplish global regulatory programs. In Caulobacter crescentus, a cascade of essential master regulators supervises the correct and sequential activation of DNA replication, cell division, and development of different cell types. Among them, the response regulator CtrA plays a crucial role coordinating all those functions. Here, for the first time, we describe the role of a novel factor named CcnA (cell cycle noncoding RNA A), a cell cycle-regulated noncoding RNA (ncRNA) located at the origin of replication, presumably activated by CtrA, and responsible for the accumulation of CtrA itself. In addition, CcnA may be also involved in the inhibition of translation of the S-phase regulator, GcrA, by interacting with its 5' untranslated region (5' UTR). Performing in vitro experiments and mutagenesis, we propose a mechanism of action of CcnA based on liberation (ctrA) or sequestration (gcrA) of their ribosome-binding site (RBS). Finally, its role may be conserved in other alphaproteobacterial species, such as Sinorhizobium meliloti, representing indeed a potentially conserved process modulating cell cycle in Caulobacterales and Rhizobiales.

HprSR Is a Reactive Chlorine Species-Sensing, Two-Component System in Escherichia coli.

Two-component systems (TCS) are signaling pathways that allow bacterial cells to sense, respond to, and adapt to fluctuating environments. Among the classical TCS of Escherichia coli, HprSR has recently been shown to be involved in the regulation of msrPQ, which encodes the periplasmic methionine sulfoxide reductase system. In this study, we demonstrated that hypochlorous acid (HOCl) induces the expression of msrPQ in an HprSR-dependent manner, whereas H2O2, NO, and paraquat (a superoxide generator) do not. Therefore, HprS appears to be an HOCl-sensing histidine kinase. Using a directed mutagenesis approach, we showed that Met residues located in the periplasmic loop of HprS are important for its activity: we provide evidence that as HOCl preferentially oxidizes Met residues, HprS could be activated via the reversible oxidation of its methionine residues, meaning that MsrPQ plays a role in switching HprSR off. We propose that the activation of HprS by HOCl could occur through a Met redox switch. HprSR appears to be the first characterized TCS able to detect reactive chlorine species (RCS) in E. coli. This study represents an important step toward understanding the mechanisms of RCS resistance in prokaryotes. IMPORTANCE Understanding how bacteria respond to oxidative stress at the molecular level is crucial in the fight against pathogens. HOCl is one of the most potent industrial and physiological microbicidal oxidants. Therefore, bacteria have developed counterstrategies to survive HOCl-induced stress. Over the last decade, important insights into these bacterial protection factors have been obtained. Our work establishes HprSR as a reactive chlorine species-sensing, two-component system in Escherichia coli MG1655, which regulates the expression of msrPQ, two genes encoding, a repair system for HOCl-oxidized proteins. Moreover, we provide evidence suggesting that HOCl could activate HprS through a methionine redox switch.

A Tad-like apparatus is required for contact-dependent prey killing in predatory social bacteria.

Myxococcus xanthus, a soil bacterium, predates collectively using motility to invade prey colonies. Prey lysis is mostly thought to rely on secreted factors, cocktails of antibiotics and enzymes, and direct contact with Myxococcus cells. In this study, we show that on surfaces the coupling of A-motility and contact-dependent killing is the central predatory mechanism driving effective prey colony invasion and consumption. At the molecular level, contact-dependent killing involves a newly discovered type IV filament-like machinery (Kil) that both promotes motility arrest and prey cell plasmolysis. In this process, Kil proteins assemble at the predator-prey contact site, suggesting that they allow tight contact with prey cells for their intoxication. Kil-like systems form a new class of Tad-like machineries in predatory bacteria, suggesting a conserved function in predator-prey interactions. This study further reveals a novel cell-cell interaction function for bacterial pili-like assemblages.

Bacterial Genetic Approach to the Study of Reactive Oxygen Species Production in Galleria mellonella During Salmonella Infection.

Over the last decade, an increasing number of reports presented Galleria mellonella larvae as an important model to study host-pathogen interactions. Coherently, increasing information became available about molecular mechanisms used by this host to cope with microbial infections but few of them dealt with oxidative stress. In this work, we addressed the role of reactive oxygen species (ROS) produced by the immune system of G. mellonella to resist against Salmonella enterica, an intracellular pathogen responsible for a wide range of infections. We confirmed that Salmonella was pathogen for G. mellonella and showed that it had to reach a minimal bacterial load within the hemolymph to kill the larvae. ROS production by G. mellonella was revealed by the virulence defects of Salmonella mutants lacking catalases/peroxiredoxins or cytoplasmic superoxide dismutases, both strains being highly sensitive to these oxidants. Finally, we used bacterial transcriptional fusions to demonstrate that hydrogen peroxide (H2O2) was produced in the hemolymph of Galleria during infection and sensed by S. enterica. In line with this observation, the H2O2-dependent regulator OxyR was found to be required for bacterial virulence in the larvae. These results led us to conclude that ROS production is an important mechanism used by G. mellonella to counteract bacterial infections and validate this host as a relevant model to study host-pathogen interactions.

OrpR is a σ54 -dependent activator using an iron-sulfur cluster for redox sensing in Desulfovibrio vulgaris Hildenborough.

Enhancer binding proteins (EBPs) are key players of σ54 -regulation that control transcription in response to environmental signals. In the anaerobic microorganism Desulfovibrio vulgaris Hildenborough (DvH), orp operons have been previously shown to be coregulated by σ54 -RNA polymerase, the integration host factor IHF and a cognate EBP, OrpR. In this study, ChIP-seq experiments indicated that the OrpR regulon consists of only the two divergent orp operons. In vivo data revealed that (i) OrpR is absolutely required for orp operons transcription, (ii) under anaerobic conditions, OrpR binds on the two dedicated DNA binding sites and leads to high expression levels of the orp operons, (iii) increasing the redox potential of the medium leads to a drastic down-regulation of the orp operons expression. Moreover, combining functional and biophysical studies on the anaerobically purified OrpR leads us to propose that OrpR senses redox potential variations via a redox-sensitive [4Fe-4S]2+ cluster in the sensory PAS domain. Overall, the study herein presents the first characterization of a new Fe-S redox regulator belonging to the σ54 -dependent transcriptional regulator family probably advantageously selected by cells adapted to the anaerobic lifestyle to monitor redox stress conditions.

Modulation of bacterial multicellularity via spatio-specific polysaccharide secretion.

The development of multicellularity is a key evolutionary transition allowing for differentiation of physiological functions across a cell population that confers survival benefits; among unicellular bacteria, this can lead to complex developmental behaviors and the formation of higher-order community structures. Herein, we demonstrate that in the social δ-proteobacterium Myxococcus xanthus, the secretion of a novel biosurfactant polysaccharide (BPS) is spatially modulated within communities, mediating swarm migration as well as the formation of multicellular swarm biofilms and fruiting bodies. BPS is a type IV pilus (T4P)-inhibited acidic polymer built of randomly acetylated ß-linked tetrasaccharide repeats. Both BPS and exopolysaccharide (EPS) are produced by dedicated Wzx/Wzy-dependent polysaccharide-assembly pathways distinct from that responsible for spore-coat assembly. While EPS is preferentially produced at the lower-density swarm periphery, BPS production is favored in the higher-density swarm interior; this is consistent with the former being known to stimulate T4P retraction needed for community expansion and a function for the latter in promoting initial cell dispersal. Together, these data reveal the central role of secreted polysaccharides in the intricate behaviors coordinating bacterial multicellularity.

Author Correction: New paradigms for understanding and step changes in treating active and chronic, persistent apicomplexan infections.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Isolation, characterization, and genome insights into an anaerobic sulfidogenic Tissierella bacterium from Cu-bearing coins.

Recent reports on antimicrobial effects of metallic Cu prompted this study of anaerobic microbial communities on copper surfaces. Widely circulating copper-containing coinage was used as a potential source for microorganisms that had had human contact and were tolerant to copper. This study reports on the isolation, characterization, and genome of an anaerobic sulfidogenic Tissierella sp. P1from copper-containing brass coinage. Dissimilatory (bi)sulfite reductase dsrAB present in strain P1 genome and the visible absorbance around 630 nm in the cells suggested the presence of a desulfoviridin-type protein. However, the sulfate reduction rate measurements with 35SO42- did not confirm the dissimilatory sulfate reduction by the strain. The P1 genome lacks APS reductase, sulfate adenylyltransferase, DsrC, and DsrMK necessary for dissimilatory sulfate reduction. The isolate produced up to 0.79 mM H2S during growth, possibly due to cysteine synthase (CysK) and/or cysteine desulfhydrase (CdsH) activities, encoded in the genome. The strain can tolerate up to 2.4 mM Cu2+(150 mg/l) in liquid medium, shows affinity to metallic copper, and can survive on copper-containing coins up to three days under ambient air and dry conditions. The genome sequence of strain P1 contained cutC, encoding a copper resistance protein, which distinguishes it from all other Tissierella strains with published genomes.

Obligate sugar oxidation in Mesotoga spp., phylum Thermotogae, in the presence of either elemental sulfur or hydrogenotrophic sulfate-reducers as electron acceptor.

Mesotoga prima strain PhosAc3 is a mesophilic representative of the phylum Thermotogae comprising only fermentative bacteria so far. We show that while unable to ferment glucose, this bacterium is able to couple its oxidation to reduction of elemental sulfur. We demonstrate furthermore that M. prima strain PhosAc3 as well as M. prima strain MesG1 and Mesotoga infera are able to grow in syntrophic association with sulfate-reducing bacteria (SRB) acting as hydrogen scavengers through interspecies hydrogen transfer. Hydrogen production was higher in M. prima strain PhosAc3 cells co-cultured with SRB than in cells cultured alone in the presence of elemental sulfur. We propose that the efficient sugar-oxidizing metabolism by M. prima strain PhosAc3 in syntrophic association with a hydrogenotrophic sulfate-reducing bacterium can be extrapolated to all members of the Mesotoga genus. Genome comparison of Thermotogae members suggests that the metabolic difference between Mesotoga and Thermotoga species (sugar oxidation versus fermentation) is mainly due to the absence of the bifurcating [FeFe]-hydrogenase in the former. Such an obligate oxidative process for using sugars, unusual within prokaryotes, is the first reported within the Thermotogae. It is hypothesized to be of primary ecological importance for growth of Mesotoga spp. in the environments that they inhabit.

Biochemical Function, Molecular Structure and Evolution of an Atypical Thioredoxin Reductase from Desulfovibrio vulgaris.

Thioredoxin reductase (TR) regulates the intracellular redox environment by reducing thioredoxin (Trx). In anaerobes, recent findings indicate that the Trx redox network is implicated in the global redox regulation of metabolism but also actively participates in protecting cells against O2. In the anaerobe Desulfovibrio vulgaris Hildenborough (DvH), there is an intriguing redundancy of the Trx system which includes a classical system using NADPH as electron source, a non-canonical system using NADH and an isolated TR (DvTRi). The functionality of DvTRi was questioned due to its lack of reactivity with DvTrxs. Structural analysis shows that DvTRi is a NAD(P)H-independent TR but its reducer needs still to be identified. Moreover, DvTRi reduced by an artificial electron source is able to reduce in turn DvTrx1 and complexation experiments demonstrate a direct interaction between DvTRi and DvTrx1. The deletion mutant tri exhibits a higher sensitivity to disulfide stress and the gene tri is upregulated by O2 exposure. Having DvTRi in addition to DvTR1 as electron source for reducing DvTrx1 must be an asset to combat oxidative stress. Large-scale phylogenomics analyses show that TRi homologs are confined within the anaerobes. All TRi proteins displayed a conserved TQ/NGK motif instead of the HRRD motif, which is selective for the binding of the 2'-phosphate group of NADPH. The evolutionary history of TRs indicates that tr1 is the common gene ancestor in prokaryotes, affected by both gene duplications and horizontal gene events, therefore leading to the appearance of TRi through subfunctionalization over the evolutionary time.

New paradigms for understanding and step changes in treating active and chronic, persistent apicomplexan infections.

Toxoplasma gondii, the most common parasitic infection of human brain and eye, persists across lifetimes, can progressively damage sight, and is currently incurable. New, curative medicines are needed urgently. Herein, we develop novel models to facilitate drug development: EGS strain T. gondii forms cysts in vitro that induce oocysts in cats, the gold standard criterion for cysts. These cysts highly express cytochrome b. Using these models, we envisioned, and then created, novel 4-(1H)-quinolone scaffolds that target the cytochrome bc1 complex Qi site, of which, a substituted 5,6,7,8-tetrahydroquinolin-4-one inhibits active infection (IC50, 30 nM) and cysts (IC50, 4 µM) in vitro, and in vivo (25 mg/kg), and drug resistant Plasmodium falciparum (IC50, <30 nM), with clinically relevant synergy. Mutant yeast and co-crystallographic studies demonstrate binding to the bc1 complex Qi site. Our results have direct impact on improving outcomes for those with toxoplasmosis, malaria, and ~2 billion persons chronically infected with encysted bradyzoites.

The primary pathway for lactate oxidation in Desulfovibrio vulgaris.

The ability to respire sulfate linked to lactate oxidation is a key metabolic signature of the Desulfovibrio genus. Lactate oxidation by these incomplete oxidizers generates reductants through lactate dehydrogenase (LDH) and pyruvate-ferredoxin oxidoreductase (PFOR), with the latter catalyzing pyruvate conversion into acetyl-CoA. Acetyl-CoA is the source of substrate-level phosphorylation through the production of ATP. Here, we show that these crucial steps are performed by enzymes encoded by a nonacistronic transcriptional unit named now as operon luo (for lactate utilization operon). Using a combination of genetic and biochemical techniques, we assigned a physiological role to the operon genes DVU3027-28 and DVU3032-33. The growth of mutant Δ26-28 was highly disrupted on D-lactate, whereas the growth of mutant Δ32-33 was slower on L-lactate, which could be related to a decrease in the activity of D-lactate or L-lactate oxidase in the corresponding mutants. The DVU3027-28 and DVU3032-33 genes thus encode functional D-LDH and L-LDH enzymes, respectively. Scanning of the genome for lactate utilization revealed several lactate permease and dehydrogenase homologs. However, transcriptional compensation was not observed in any of the mutants except for lactate permease. Although there is a high degree of redundancy for lactate oxidase, it is not functionally efficient in LDH mutants. This result could be related to the identification of several operon enzymes, including LDHs, in the PFOR activity bands, suggesting the occurrence of a lactate-oxidizing supermolecular structure that can optimize the performance of lactate utilization in Desulfovibrio species.

Growth of the obligate anaerobe Desulfovibrio vulgaris Hildenborough under continuous low oxygen concentration sparging: impact of the membrane-bound oxygen reductases.

Although obligate anaerobe, the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough (DvH) exhibits high aerotolerance that involves several enzymatic systems, including two membrane-bound oxygen reductases, a bd-quinol oxidase and a cc(b/o)o3 cytochrome oxidase. Effect of constant low oxygen concentration on growth and morphology of the wild-type, single (Δbd, Δcox) and double deletion (Δcoxbd) mutant strains of the genes encoding these oxygen reductases was studied. When both wild-type and deletion mutant strains were cultured in lactate/sulfate medium under constant 0.02% O2 sparging, they were able to grow but the final biomasses and the growth yield were lower than that obtained under anaerobic conditions. At the end of the growth, lactate was not completely consumed and when conditions were then switched to anaerobic, growth resumed. Time-lapse microscopy revealed that a large majority of the cells were then able to divide (over 97%) but the time to recover a complete division event was longer for single deletion mutant Δbd than for the three other strains. Determination of the molar growth yields on lactate suggested that a part of the energy gained from lactate oxidation was derived toward cells protection/repairing against oxidative conditions rather than biosynthesis, and that this part was higher in the single deletion mutant Δbd and, to a lesser extent, Δcox strains. Our data show that when DvH encounters oxidative conditions, it is able to stop growing and to rapidly resume growing when conditions are switched to anaerobic, suggesting that it enters active dormancy sate under oxidative conditions. We propose that the pyruvate-ferredoxin oxidoreductase (PFOR) plays a central role in this phenomenon by reversibly switching from an oxidative-sensitive fully active state to an oxidative-insensitive inactive state. The oxygen reductases, and especially the bd-quinol oxidase, would have a crucial function by maintaining reducing conditions that permit PFOR to stay in its active state.

Additive effect of nuclear and mitochondrial mutations in a patient with mitochondrial encephalomyopathy.

We describe the case of a woman in whom combination of a mitochondrial (MT-CYB) and a nuclear (SDHB) mutation was associated with clinical and metabolic features suggestive of a mitochondrial disorder. The mutations impaired overall energy metabolism in the patient's muscle and fibroblasts and increased cellular susceptibility to oxidative stress. To clarify the contribution of each mutation to the phenotype, mutant yeast strains were generated. A significant defect in strains carrying the Sdh2 mutation, either alone or in combination with the cytb variant, was observed. Our data suggest that the SDHB mutation was causative of the mitochondrial disorder in our patient with a possible cumulative contribution of the MT-CYB variant. To our knowledge, this is the first association of bi-genomic variants in the mtDNA and in a nuclear gene encoding a subunit of complex II.

An oxygen reduction chain in the hyperthermophilic anaerobe Thermotoga maritima highlights horizontal gene transfer between Thermococcales and Thermotogales.

The hyperthermophile Thermotoga maritima, although strictly anaerobic, is able to grow in the presence of low amounts of O(2). Here, we show that this bacterium consumes O(2) via a three-partner chain involving an NADH oxidoreductase (NRO), a rubredoxin (Rd) and a flavo-diiron protein (FprA) (locus tags: TM_0754, TM_0659 and TM_0755, respectively). In vitro experiments showed that the NADH-dependent O(2) consumption rate was 881.9 (± 106.7) mol O(2) consumed min(-1) per mol of FprA at 37°C and that water was the main end-product of the reaction. We propose that this O(2) reduction chain plays a central role in the O(2) tolerance of T. maritima. Phylogenetic analyses suggest that the genes coding for these three components were acquired by an ancestor of Thermotogales from an ancestor of Thermococcales via a single gene transfer. This event likely also involved two ROS scavenging enzymes (neelaredoxin and rubrerythrin) that are encoded by genes clustered with those coding for FprA, NRO and Rd in the ancestor of Thermococcales. Such genomic organization would have provided the ancestor of Thermotogales with a complete set of enzymes dedicated to O(2)-toxicity defence. Beside Thermotogales and Thermococcales, horizontal gene transfers have played a major role in disseminating these enzymes within the hyperthermophilic anaerobic prokaryotic communities, allowing them to cope with fluctuating oxidative conditions that exist in situ.

Study of the thiol/disulfide redox systems of the anaerobe Desulfovibrio vulgaris points out pyruvate:ferredoxin oxidoreductase as a new target for thioredoxin 1.

Sulfate reducers have developed a multifaceted adaptative strategy to survive against oxidative stresses. Along with this oxidative stress response, we recently characterized an elegant reversible disulfide bond-dependent protective mechanism in the pyruvate:ferredoxin oxidoreductase (PFOR) of various Desulfovibrio species. Here, we searched for thiol redox systems involved in this mechanism. Using thiol fluorescent labeling, we show that glutathione is not the major thiol/disulfide balance-controlling compound in four different Desulfovibrio species and that no other plentiful low molecular weight thiol can be detected. Enzymatic analyses of two thioredoxins (Trxs) and three thioredoxin reductases allow us to propose the existence of two independent Trx systems in Desulfovibrio vulgaris Hildenborough (DvH). The TR1/Trx1 system corresponds to the typical bacterial Trx system. We measured a TR1 apparent K(m) value for Trx1 of 8.9 µM. Moreover, our results showed that activity of TR1 was NADPH-dependent. The second system named TR3/Trx3 corresponds to an unconventional Trx system as TR3 used preferentially NADH (K(m) for NADPH, 743 µM; K(m) for NADH, 5.6 µM), and Trx3 was unable to reduce insulin. The K(m) value of TR3 for Trx3 was 1.12 µM. In vitro experiments demonstrated that the TR1/Trx1 system was the only one able to reactivate the oxygen-protected form of Desulfovibrio africanus PFOR. Moreover, ex vivo pulldown assays using the mutant Trx1(C33S) as bait allowed us to capture PFOR from the DvH extract. Altogether, these data demonstrate that PFOR is a new target for Trx1, which is probably involved in the protective switch mechanism of the enzyme.

Is cytochrome b glutamic acid 272 a quinol binding residue in the bc1 complex of Saccharomyces cerevisiae?

The mitochondrial bc1 complex catalyzes the oxidation of ubiquinol and the reduction of cytochrome (cyt) c coupled to a vectorial translocation of protons across the membrane. On the basis of the three-dimensional structures of the bc1 complex in the presence of the inhibitor stigmatellin, it was assumed that the substrate quinol binding involves the cyt b glutamate residue E272 and the histidine 181 on the Rieske protein. Although extensive mutagenesis of glutamate E272 has been carried out, different experimental results were recently obtained, and different conclusions were drawn to explain its role in the bifurcated electron/proton transfer at the QO site. This residue is not totally conserved during evolution. We show in this study that replacement of E272 with apolar residues proline and valine naturally present in some organisms did not abolish the bc1 activity, although it slowed down the kinetics of electron transfer. The Km value for the binding of the substrate quinol was not modified, and the EPR data showed that the quinone/quinol binding still occurred in the mutants. Binding of stigmatellin was retained; however, mutations E272P,V induced resistance toward the QO site inhibitor myxothiazol. The pH dependence of the bc1 activity was not modified in the absence of the glutamate E272. Our results suggest that this residue may not be involved in direct substrate binding or in its direct deprotonation. Revertants were selected from the respiratory deficient mutant E272P. The observed suppressor mutations introduced polar residues serine and threonine at position 272. The data lead us to suggest that E272 may be involved in a later step on the proton exit pathway via the interaction with a water molecule.