Physical and functional interplay between PCNA DNA clamp and Mre11-Rad50 complex from the archaeon Pyrococcus furiosus.

Several archaeal species prevalent in extreme environments are particularly exposed to factors likely to cause DNA damages. These include hyperthermophilic archaea (HA), living at temperatures >70°C, which arguably have efficient strategies and robust genome guardians to repair DNA damage threatening their genome integrity. In contrast to Eukarya and other archaea, homologous recombination appears to be a vital pathway in HA, and the Mre11-Rad50 complex exerts a broad influence on the initiation of this DNA damage response process. In a previous study, we identified a physical association between the Proliferating Cell Nuclear Antigen (PCNA) and the Mre11-Rad50 (MR) complex. Here, by performing co-immunoprecipitation and SPR analyses, we identified a short motif in the C- terminal portion of Pyrococcus furiosus Mre11 involved in the interaction with PCNA. Through this work, we revealed a PCNA-interaction motif corresponding to a variation on the PIP motif theme which is conserved among Mre11 sequences of Thermococcale species. Additionally, we demonstrated functional interplay in vitro between P. furiosus PCNA and MR enzymatic functions in the DNA end resection process. At physiological ionic strength, PCNA stimulates MR nuclease activities for DNA end resection and promotes an endonucleolytic incision proximal to the 5' strand of double strand DNA break.

Yarrowia lipolytica AAL genes are involved in peroxisomal fatty acid activation.

In yeast, ß-oxidation of fatty acids (FAs) essentially takes place in peroxisomes, and FA activation must precede FA oxidation. In Saccharomyces cerevisiae, a single fatty-acyl­CoA-synthetase, ScFaa2p, mediates peroxisomal FA activation. We have previously shown that this reaction also exists in the oleaginous yeast Yarrowia lipolytica; however, the protein involved in this process remains unknown. Here, we found that proteins, named Aal proteins (Acyl/Aryl-CoA-ligases), resembling the 4-coumarate­CoA-ligase-like enzymes found in plants are involved in peroxisomal FA activation in Y. lipolytica; Y. lipolytica has 10 AAL genes, eight of which are upregulated by oleate. All the Aal proteins contain a PTS1-type peroxisomal targeting sequence (A/SKL), suggesting a peroxisomal localization. The function of the Aal proteins was analyzed using the faa1Δant1Δ mutant strain, which demonstrates neither cytoplasmic FA activation (direct result of FAA1 deletion) nor peroxisomal FA activation (indirect result of ANT1 deletion, a gene coding an ATP transporter). This strain is thus highly sensitive to external FA levels and unable to store external FAs in lipid bodies (LBs). Whereas the overexpression of (cytoplasmic) AAL1ΔPTS1 was able to partially complement the growth defect observed in the faa1Δant1Δ mutant on short-, medium- and long-chain FA media, the presence of Aal2p to Aal10p only allowed growth on the short-chain FA medium. Additionally, partial LB formation was observed in the oleate medium for strains overexpressing Aal1ΔPTS1p, Aal4ΔPTS1p, Aal7ΔPTS1p, and Aal8ΔPTS1p. Finally, an analysis of the FA content of cells grown in the oleate medium suggested that Aal4p and Aal6p present substrate specificity for C16:1 and/or C18:0.

Transforming sugars into fat - lipid biosynthesis using different sugars in Yarrowia lipolytica.

In an era of ever-increasing energy demands, a promising technology is being developed: the use of oleaginous microorganisms such as Yarrowia lipolytica to convert waste materials into biofuels. Here, we constructed two Y. lipolytica strains that displayed both increased lipid accumulation and more efficient use of biomass-derived sugars, including glucose, fructose, galactose and inulin. The first strain, Y. lipolytica YLZ150, was derived from the French wild-type strain W29. It had inhibited triacylglycerol mobilization (∆tgl4) and ß-oxidation (∆pox1-6), and it overexpressed GPD1, DGA2, HXK1, the native Leloir pathway, SUC2 from Saccharomyces cerevisiae and INU1 from Kluyveromyces marxianus. The second strain, Y. lipolytica Y4779, was derived from the Polish A-101 strain. It had inhibited ß-oxidation (∆mfe2) and overexpressed GPD1, DGA1, HXK1, YHT3, SUC2 and INU1. In the first experiment, strain YLZ150 was batch-cultured in media containing different hexoses; the highest values for lipid concentration and yield of lipids from the substrate were obtained using fructose (20.3 g dm-3 and 0.14 g g-1 , respectively). In the second experiment, we grew the two strains in fed-batch cultures to examine lipid biosynthesis from inulin (a fructose polymer). For Y4779, the lipid concentration was 10.3 g dm-3 and the yield of lipids from substrate was 0.07 g g-1 ; in contrast, for YLZ150, these values were 24 g dm-3 and 0.16 g g-1 , respectively. The YLZ150 strain is thus able to efficiently exploit glucose, fructose, galactose, sucrose and inulin for lipid biosynthesis. Copyright © 2017 John Wiley & Sons, Ltd.

Using a vector pool containing variable-strength promoters to optimize protein production in Yarrowia lipolytica.

BACKGROUND: The yeast Yarrowia lipolytica is an increasingly common biofactory. To enhance protein expression, several promoters have been developed, including the constitutive TEF promoter, the inducible POX2 promotor, and the hybrid hp4d promoter. Recently, new hp4d-inspired promoters have been created that couple various numbers of UAS1 tandem elements with the minimal LEU2 promoter or the TEF promoter. Three different protein-secretion signaling sequences can be used: preLip2, preXpr2, and preSuc2. RESULTS: To our knowledge, our study is the first to use a set of vectors with promoters of variable strength to produce proteins of industrial interest. We used the more conventional TEF and hp4d promoters along with five new hybrid promoters: 2UAS1-pTEF, 3UAS1-pTEF, 4UAS1-pTEF, 8UAS1-pTEF, and hp8d. We compared the production of RedStar2, glucoamylase, and xylanase C when strains were grown on three media. As expected, levels of RedStar2 and glucoamylase were greatest in the strain with the 8UAS1-pTEF promoter, which was stronger. However, surprisingly, the 2UAS1-pTEF promoter was associated with the greatest xylanase C production and activity. This finding underscored that stronger promoters are not always better when it comes to protein production. We therefore developed a method for easily identifying the best promoter for a given protein of interest. In this gateway method, genes for YFP and α-amylase were transferred into a pool of vectors containing different promoters and gene expression was then analyzed. We observed that, in most cases, protein production and activity were correlated with promoter strength, although this pattern was protein dependent. CONCLUSIONS: Protein expression depends on more than just promoter strength. Indeed, promoter suitability appears to be protein dependent; in some cases, optimal expression and activity was obtained using a weaker promoter. We showed that using a vector pool containing promoters of variable strength can be a powerful tool for rapidly identifying the best producer for a given protein of interest.

Unraveling fatty acid transport and activation mechanisms in Yarrowia lipolytica.

Fatty acid (FA) transport and activation have been extensively studied in the model yeast species Saccharomyces cerevisiae but have rarely been examined in oleaginous yeasts, such as Yarrowia lipolytica. Because the latter begins to be used in biodiesel production, understanding its FA transport and activation mechanisms is essential. We found that Y. lipolytica has FA transport and activation proteins similar to those of S. cerevisiae (Faa1p, Pxa1p, Pxa2p, Ant1p) but mechanism of FA peroxisomal transport and activation differs greatly with that of S. cerevisiae. While the ScPxa1p/ScPxa2p heterodimer is essential for growth on long-chain FAs, ΔYlpxa1 ΔYlpxa2 is not impaired for growth on FAs. Meanwhile, ScAnt1p and YlAnt1p are both essential for yeast growth on medium-chain FAs, suggesting they function similarly. Interestingly, we found that the ΔYlpxa1 ΔYlpxa2 ΔYlant1 mutant was unable to grow on short-, medium-, or long-chain FAs, suggesting that YlPxa1p, YlPxa2p, and YlAnt1p belong to two different FA degradation pathways. We also found that YlFaa1p is involved in FA storage in lipid bodies and that FA remobilization largely depended on YlFat1p, YlPxa1p and YlPxa2p. This study is the first to comprehensively examine FA intracellular transport and activation in oleaginous yeast.

Analysis of ATP-citrate lyase and malic enzyme mutants of Yarrowia lipolytica points out the importance of mannitol metabolism in fatty acid synthesis.

The role of the two key enzymes of fatty acid (FA) synthesis, ATP-citrate lyase (Acl) and malic enzyme (Mae), was analyzed in the oleaginous yeast Yarrowia lipolytica. In most oleaginous yeasts, Acl and Mae are proposed to provide, respectively, acetyl-CoA and NADPH for FA synthesis. Acl was mainly studied at the biochemical level but no strain depleted for this enzyme was analyzed in oleaginous microorganisms. On the other hand the role of Mae in FA synthesis in Y. lipolytica remains unclear since it was proposed to be a mitochondrial NAD(H)-dependent enzyme and not a cytosolic NADP(H)-dependent enzyme. In this study, we analyzed for the first time strains inactivated for corresponding genes. Inactivation of ACL1 decreases FA synthesis by 60 to 80%, confirming its essential role in FA synthesis in Y. lipolytica. Conversely, inactivation of MAE1 has no effects on FA synthesis, except in a FA overaccumulating strain where it improves FA synthesis by 35%. This result definitively excludes Mae as a major key enzyme for FA synthesis in Y. lipolytica. During the analysis of both mutants, we observed a negative correlation between FA and mannitol level. As mannitol and FA pathways may compete for carbon storage, we inactivated YlSDR, encoding a mannitol dehydrogenase converting fructose and NADPH into mannitol and NADP+. The FA content of the resulting mutant was improved by 60% during growth on fructose, demonstrating that mannitol metabolism may modulate FA synthesis in Y. lipolytica.

Combining metabolic engineering and process optimization to improve production and secretion of fatty acids.

Microbial oils are sustainable alternatives to petroleum for the production of chemicals and fuels. Oleaginous yeasts are promising source of oils and Yarrowia lipolytica is the most studied and engineered one. Nonetheless the commercial production of biolipids is so far limited to high value products due to the elevated production and extraction costs. In order to contribute to overcoming these limitations we exploited the possibility of secreting lipids to the culture broth, uncoupling production and biomass formation and facilitating the extraction. We therefore considered two synthetic approaches, Strategy I where fatty acids are produced by enhancing the flux through neutral lipid formation, as typically occurs in eukaryotic systems and Strategy II where the bacterial system to produce free fatty acids is mimicked. The engineered strains, in a coupled fermentation and extraction process using alkanes, secreted the highest titer of lipids described so far, with a content of 120% of DCW.

Truncation of Gal4p explains the inactivation of the GAL/MEL regulon in both Saccharomyces bayanus and some Saccharomyces cerevisiae wine strains.

In the past, the galactose-negative (Gal(-)) phenotype was a key physiological character used to distinguish Saccharomyces bayanus from S. cerevisiae In this work, we investigated the inactivation of GAL gene networks in S. bayanus, which is an S. uvarum/S. eubayanus hybrid, and in S. cerevisiae wine strains erroneously labelled 'S. bayanus'. We made an inventory of their GAL genes using genomes that were either available publicly, re-sequenced by us, or assembled from public data and completed with targeted sequencing. In the S. eubayanus/S. uvarum CBS 380(T) hybrid, the GAL/MEL network is composed of genes from both parents: from S. uvarum, an otherwise complete set that lacks GAL4, and from S. eubayanus, a truncated version of GAL4 and an additional copy of GAL3 and GAL80 Similarly, two different truncated GAL4 alleles were found in S. cerevisiae wine strains EC1118 and LalvinQA23. The lack of GAL4 activity in these strains was corrected by introducing a full-length copy of S. cerevisiae GAL4 on a CEN4/ARS plasmid. Transformation with this plasmid restored galactose utilisation in Gal(-) strains, and melibiose fermentation in strain CBS 380(T) The melibiose fermentation phenotype, formerly regarded as characteristic of S. uvarum, turned out to be widespread among Saccharomyces species.

Role of Pex11p in Lipid Homeostasis in Yarrowia lipolytica.

Peroxisomes are essential organelles in the cells of most eukaryotes, from yeasts to mammals. Their role in ß-oxidation is particularly essential in yeasts; for example, in Saccharomyces cerevisiae, fatty acid oxidation takes place solely in peroxisomes. In this species, peroxisome biogenesis occurs when lipids are present in the culture medium, and it involves the Pex11p protein family: ScPex11p, ScPex25p, ScPex27p, and ScPex34p. Yarrowia lipolytica has three Pex11p homologues, which are YALI0C04092p (YlPex11p), YALI0C04565p (YlPex11C), and YALI0D25498p (Pex11/25p). We found that these genes are regulated by oleic acid, and as has been observed in other organisms, YlPEX11 deletion generated giant peroxisomes when mutant yeast were grown in oleic acid medium. Moreover, ΔYlpex11 was unable to grow on fatty acid medium and showed extreme dose-dependent sensitivity to oleic acid. Indeed, when the strain was grown in minimum medium with 0.5% glucose and 3% oleic acid, lipid body lysis and cell death were observed. Cell death and lipid body lysis may be partially explained by an imbalance in the expression of the genes involved in lipid storage, namely, DGA1, DGA2, and LRO1, as well as that of TGL4, which is involved in lipid remobilization. TGL4 deletion and DGA2 overexpression resulted in decreased oleic acid sensitivity and delayed cell death of ΔYlpex11, which probably stemmed from the release of free fatty acids into the cytoplasm. All these results show that YlPex11p plays an important role in lipid homeostasis in Y. lipolytica.

Radiation response in Deinococcus deserti: IrrE is a metalloprotease that cleaves repressor protein DdrO.

Deinococcus bacteria are famous for their extreme radiation tolerance. The IrrE protein was shown to be essential for radiation tolerance and, in an unelucidated manner, for induction of a number of genes in response to radiation, including recA and other DNA repair genes. Earlier studies indicated that IrrE could be a zinc peptidase, but proteolytic activity was not demonstrated. Here, using several in vivo and in vitro experiments, IrrE from Deinococcus deserti was found to interact with DdrO, a predicted regulator encoded by a radiation-induced gene that is, like irrE, highly conserved in Deinococcus. Moreover, IrrE was found to cleave DdrO in vitro and when the proteins were coexpressed in Escherichia coli. This cleavage was not observed in the presence of metal chelator EDTA or when IrrE contains a mutation in the conserved active-site motif of metallopeptidases. In D. deserti, IrrE-dependent cleavage of DdrO was observed after exposure to radiation. Furthermore, DdrO-dependent repression of the promoter of a radiation-induced gene was shown. These results demonstrate that IrrE is a metalloprotease and we propose that IrrE-mediated cleavage inactivates repressor protein DdrO, leading to transcriptional induction of various genes required for repair and survival after exposure of Deinococcus to radiation.

The abundant and essential HU proteins in Deinococcus deserti and Deinococcus radiodurans are translated from leaderless mRNA.

HU proteins have an important architectural role in nucleoid organization in bacteria. Compared with HU of many bacteria, HU proteins from Deinococcus species possess an N-terminal lysine-rich extension similar to the eukaryotic histone H1 C-terminal domain involved in DNA compaction. The single HU gene in Deinococcus radiodurans, encoding DrHU, is required for nucleoid compaction and cell viability. Deinococcus deserti contains three expressed HU genes, encoding DdHU1, DdHU2 and DdHU3. Here, we show that either DdHU1 or DdHU2 is essential in D. deserti. DdHU1 and DdHU2, but not DdHU3, can substitute for DrHU in D. radiodurans, indicating that DdHU3 may have a non-essential function different from DdHU1, DdHU2 and DrHU. Interestingly, the highly abundant DrHU and DdHU1 proteins, and also the less expressed DdHU2, are translated in Deinococcus from leaderless mRNAs, which lack a 5'-untranslated region and, hence, the Shine-Dalgarno sequence. Unexpectedly, cloning the DrHU or DdHU1 gene under control of a strong promoter in an expression plasmid, which results in leadered transcripts, strongly reduced the DrHU and DdHU1 protein level in D. radiodurans compared with that obtained from the natural leaderless gene. We also show that the start codon position for DrHU and DdHU1 should be reannotated, resulting in proteins that are 15 and 4 aa residues shorter than initially reported. The expression level and start codon correction were crucial for functional characterization of HU in Deinococcus.

ISDra2 transposition in Deinococcus radiodurans is downregulated by TnpB.

Transposable elements belonging to the recently identified IS200/IS605 family radically differ from classical insertion sequences in their transposition mechanism by strictly requiring single-stranded DNA substrates. This IS family includes elements encoding only the transposase (TnpA), and others, like ISDra2 from Deinococcus radiodurans, which contain a second gene, tnpB, dispensable for transposition and of unknown function to date. Here, we show that TnpB has an inhibitory effect on the excision and insertion steps of ISDra2 transposition. This inhibitory action of TnpB was maintained when ISDra2 transposition was induced by γ-irradiation of the host cells and required the integrity of its putative zinc finger motif. We also demonstrate the negative role of TnpB when ISDra2 transposition was monitored in a heterologous Escherichia coli host, indicating that TnpB-mediated inhibition does not involve Deinococcus-specific factors. TnpB therefore appears to play a regulatory role in ISDra2 transposition.

The fatty acid transport protein Fat1p is involved in the export of fatty acids from lipid bodies in Yarrowia lipolytica.

In order to live, cells need to import different molecules, such as sugars, amino acids or lipids, using transporters. In Saccharomyces cerevisiae, the ScFAT1 gene encodes the long-chain fatty acid transporter; however, the transport of fatty acids (FAs) in the oleaginous yeast Yarrowia lipolytica has not yet been studied. In contrast to what has previously been found for ΔScfat1 strains, ΔYlfat1 yeast was still able to grow on substrates containing short-, medium- or long-chain FAs. We observed a notable difference in cell lipid content between wild-type (WT) and deletion mutant strains after 24 h of culture in minimal oleate medium: in the WT strain, lipids represented 24% of cell dry weight (CDW), while they accounted for 37% of CDW in the ΔYlfat1 strain. This result indicates that YlFat1p is not involved in cell lipid uptake. Moreover, we also observed that fatty acid remobilisation was decreased in the ΔYlfat1 strain and that fluorescence-tagged YlFat1p proteins localised to the interfaces between lipid bodies, which suggests that YlFat1p may play a role in the export of FAs from lipid bodies.

Cooperation between two modes for DNA replication initiation in the archaeon Thermococcus barophilus.

The mechanisms underpinning the replication of genomic DNA have recently been challenged in Archaea. Indeed, the lack of origin of replication has no deleterious effect on growth, suggesting that replication initiation relies on homologous recombination. Recombination-dependent replication (RDR) appears to be based on the recombinase RadA, which is of absolute requirement when no initiation origins are detected. The origin of this flexibility in the initiation of replication and the extent to which it is used in nature are yet to be understood. Here, we followed the process of DNA replication throughout the growth stages of Thermococcus barophilus. We combined deep sequencing and genetics to elucidate the dynamics of oriC utilization according to growth phases. We discovered that in T. barophilus, the use of oriC diminishes from the lag to the middle of the log phase, and subsequently increases gradually upon entering the stationary phase. Although oriC demonstrates no indispensability, RadA does exhibit essentiality. Notably, a knockdown mutant strain provides confirmation of the pivotal role of RadA in RDR for the first time. Thus, we demonstrate the existence of a tight combination between oriC utilization and homologous recombination to initiate DNA replication along the growth phases. Overall, this study demonstrates how diverse physiological states can influence the initiation of DNA replication, offering insights into how environmental sensing might impact this fundamental mechanism of life. IMPORTANCE: Replication of DNA is highly important in all organisms. It initiates at a specific locus called ori, which serves as the binding site for scaffold proteins-either Cdc6 or DnaA-depending on the domain of life. However, recent studies have shown that the Archaea, Haloferax volcanii and Thermococcus kodakarensis could subsist without ori. Recombination-dependent replication (RDR), via the recombinase RadA, is the mechanism that uses homologous recombination to initiate DNA replication. The extent to which ori's use is necessary in natural growth remains to be characterized. In this study, using Thermococcus barophilus, we demonstrated that DNA replication initiation relies on both oriC and RDR throughout its physiological growth, each to varying degrees depending on the phase. Notably, a knockdown RadA mutant confirmed the prominent use of RDR during the log phase. Moreover, the study of ploidy in oriC and radA mutant strains showed that the number of chromosomes per cell is a critical proxy for ensuring proper growth and cell survival.

Alliance of proteomics and genomics to unravel the specificities of Sahara bacterium Deinococcus deserti.

To better understand adaptation to harsh conditions encountered in hot arid deserts, we report the first complete genome sequence and proteome analysis of a bacterium, Deinococcus deserti VCD115, isolated from Sahara surface sand. Its genome consists of a 2.8-Mb chromosome and three large plasmids of 324 kb, 314 kb, and 396 kb. Accurate primary genome annotation of its 3,455 genes was guided by extensive proteome shotgun analysis. From the large corpus of MS/MS spectra recorded, 1,348 proteins were uncovered and semiquantified by spectral counting. Among the highly detected proteins are several orphans and Deinococcus-specific proteins of unknown function. The alliance of proteomics and genomics high-throughput techniques allowed identification of 15 unpredicted genes and, surprisingly, reversal of incorrectly predicted orientation of 11 genes. Reversal of orientation of two Deinococcus-specific radiation-induced genes, ddrC and ddrH, and identification in D. deserti of supplementary genes involved in manganese import extend our knowledge of the radiotolerance toolbox of Deinococcaceae. Additional genes involved in nutrient import and in DNA repair (i.e., two extra recA, three translesion DNA polymerases, a photolyase) were also identified and found to be expressed under standard growth conditions, and, for these DNA repair genes, after exposure of the cells to UV. The supplementary nutrient import and DNA repair genes are likely important for survival and adaptation of D. deserti to its nutrient-poor, dry, and UV-exposed extreme environment.

Mutagenic lesion bypass and two functionally different RecA proteins in Deinococcus deserti.

RecA is essential for extreme radiation tolerance in Deinococcus radiodurans. Interestingly, Sahara bacterium Deinococcus deserti has three recA genes (recA(C), recA(P1), recA(P3)) that code for two different RecA proteins (RecA(C), RecA(P)). Moreover, and in contrast to other sequenced Deinococcus species, D. deserti possesses homologues of translesion synthesis (TLS) DNA polymerases, including ImuY and DnaE2. Together with a lexA homologue, imuY and dnaE2 form a gene cluster similar to a widespread RecA/LexA-controlled mutagenesis cassette. After having developed genetic tools, we have constructed mutant strains to characterize these recA and TLS polymerase genes in D. deserti. Both RecA(C) and RecA(P) are functional and allow D. deserti to survive, and thus repair massive DNA damage, after exposure to high doses of radiation. D. deserti is mutable by UV, which requires ImuY, DnaE2 and RecA(C), but not RecA(P). RecA(C), but not RecA(P), facilitates induced expression of imuY and dnaE2 following UV exposure. We propose that the extra recA(P1) and recA(P3) genes may provide higher levels of RecA protein for efficient error-free repair of DNA damage, without further increasing error-prone lesion bypass by ImuY and DnaE2, whereas limited TLS may contribute to adaptation to harsh conditions by generating genetic variability.

Role of RadA and DNA Polymerases in Recombination-Associated DNA Synthesis in Hyperthermophilic Archaea.

Among the three domains of life, the process of homologous recombination (HR) plays a central role in the repair of double-strand DNA breaks and the restart of stalled replication forks. Curiously, main protein actors involved in the HR process appear to be essential for hyperthermophilic Archaea raising interesting questions about the role of HR in replication and repair strategies of those Archaea living in extreme conditions. One key actor of this process is the recombinase RadA, which allows the homologous strand search and provides a DNA substrate required for following DNA synthesis and restoring genetic information. DNA polymerase operation after the strand exchange step is unclear in Archaea. Working with Pyrococcus abyssi proteins, here we show that both DNA polymerases, family-B polymerase (PolB) and family-D polymerase (PolD), can take charge of processing the RadA-mediated recombination intermediates. Our results also indicate that PolD is far less efficient, as compared with PolB, to extend the invaded DNA at the displacement-loop (D-loop) substrate. These observations coincide with previous genetic analyses obtained on Thermococcus species showing that PolB is mainly involved in DNA repair without being essential probably because PolD could take over combined with additional partners.

The evolution of Jen3 proteins and their role in dicarboxylic acid transport in Yarrowia.

Jen proteins in yeast are involved in the uptake of mono/dicarboxylic acids. The Jen1 subfamily transports lactate and pyruvate, while the Jen2 subfamily transports fumarate, malate, and succinate. Yarrowia lipolytica has six JEN genes: YALI0B19470g, YALI0C15488g, YALI0C21406g, YALI0D20108g, YALI0D24607g, and YALI0E32901g. Through phylogenetic analyses, we found that these genes represent a new subfamily, Jen3 and that these three Jen subfamilies derivate from three putative ancestral genes. Reverse transcription-PCR. revealed that only four YLJEN genes are expressed and they are upregulated in the presence of lactate, pyruvate, fumarate, malate, and/or succinate, suggesting that they are able to transport these substrates. Analysis of deletion mutant strains revealed that Jen3 subfamily proteins transport fumarate, malate, and succinate. We found evidence that YALI0C15488 encodes the main transporter because its deletion was sufficient to strongly reduce or suppress growth in media containing fumarate, malate, or succinate. It appears that the other YLJEN genes play a minor role, with the exception of YALI0E32901g, which is important for malate uptake. However, the overexpression of each YLJEN gene in the sextuple-deletion mutant strain ΔYLjen1-6 revealed that all six genes are functional and have evolved to transport different substrates with varying degrees of efficacy. In addition, we found that YALI0E32901p transported succinate more efficiently in the presence of lactate or fumarate.

Identification of new genes contributing to the extreme radioresistance of Deinococcus radiodurans using a Tn5-based transposon mutant library.

Here, we have developed an extremely efficient in vivo Tn5-based mutagenesis procedure to construct a Deinococcus radiodurans insertion mutant library subsequently screened for sensitivity to genotoxic agents such as γ and UV radiations or mitomycin C. The genes inactivated in radiosensitive mutants belong to various functional categories, including DNA repair functions, stress responses, signal transduction, membrane transport, several metabolic pathways, and genes of unknown function. Interestingly, preliminary characterization of previously undescribed radiosensitive mutants suggests the contribution of cyclic di-AMP signaling in the recovery of D. radiodurans cells from genotoxic stresses, probably by modulating several pathways involved in the overall cell response. Our analyses also point out a new transcriptional regulator belonging to the GntR family, encoded by DR0265, and a predicted RNase belonging to the newly described Y family, both contributing to the extreme radioresistance of D. radiodurans. Altogether, this work has revealed new cell responses involved either directly or indirectly in repair of various cell damage and confirmed that D. radiodurans extreme radiation resistance is determined by a multiplicity of pathways acting as a complex network.

Crystal structure of the IrrE protein, a central regulator of DNA damage repair in deinococcaceae.

Deinococcaceae are famous for their extreme radioresistance. Transcriptome analysis in Deinococcus radiodurans revealed a group of genes up-regulated in response to desiccation and ionizing radiation. IrrE, a novel protein initially found in D. radiodurans, was shown to be a positive regulator of some of these genes. Deinococcus deserti irrE is able to restore radioresistance in a D. radiodurans DeltairrE mutant. The D. deserti IrrE crystal structure reveals a unique combination of three domains: one zinc peptidase-like domain, one helix-turn-helix motif and one GAF-like domain. Mutant analysis indicates that the first and third domains are critical regions for radiotolerance. In particular, mutants affected in the putative zinc-binding site are as sensitive to gamma and UV irradiation as the DeltairrE bacteria, and radioresistance is strongly decreased with the H217L mutation present in the C-terminal domain. In addition, modeling of IrrE-DNA interaction suggests that the observed IrrE structure may not bind double-stranded DNA through its central helix-turn-helix motif and that IrrE is not a classic transcriptional factor that activates gene expression by its direct binding to DNA. We propose that the putative protease activity of IrrE could be a key element of transcription enhancement and that a more classic transcription factor, possibly an IrrE substrate, would link IrrE to transcription of genes specifically involved in radioresistance.