Spodoptera, namely a semi-selective, C14:acyl-specific pgFAR and a highly selective, C16:acyl-specific pgFAR, and demonstrate that these pgFARs play a pivotal role in the formation of species-specific signals, a finding that is strongly supported by functional gene expression data. The study envisages a new area of research for disclosing evolutionary changes associated with C14- and C16-specific FARs in moth pheromone biosynthesis.Fatty acyl reductases (FARs) constitute an evolutionarily conserved gene family found in all kingdoms of life. Members of the FAR gene family play diverse roles, including seed oil synthesis, insect pheromone biosynthesis, and mammalian wax biosynthesis. In insects, FAR genes dedicated to sex pheromone biosynthesis  form a unique clade that exhibits substantial modifications in gene structure and possesses unique specificity and selectivity for fatty acyl substrates. Highly selective and semi-selective ‘single pgFARs’ produce single and multicomponent pheromone signals in bombycid, pyralid, yponomeutid and noctuid moths. An intriguing question is how a ‘single reductase’ can direct the synthesis of several fatty alcohols of various chain lengths and isomeric forms. Here, we report two active pgFARs in the pheromone gland of  Insects use sophisticated chemical communication systems, including behavior-modifying pheromones, and these systems reinforce intra- and interspecific reproductive isolation and thus play pivotal roles in the evolutionary process. The insect order Lepidoptera (moth and butterflies) contains the second largest number of species , and their richness and diversity are linked to pheromone-based communication and sophisticated chemosensory systems125781110-C18 fatty acyl moieties that are modified in the pheromone gland (PG) through the addition of functional groups, such as alcohol, acetate ester, or aldehyde. The biosynthesis of pheromone bouquets involves specialized enzymes, including fatty acyl-CoA desaturases, fatty acyl-CoA reductases (FARs), alcohol oxidases and fatty alcohol acetyltransferases7111415161714 or C16 acyls11141617Spodoptera species, utilize multicomponent C12, C14 and C16 alcohol and acetate ester pheromone blends. Of the thirty Spodoptera species, the pheromone mixtures identified from 18 species  protein database identified two candidate FARs from S. exigua that show significant identity with those of the nymphalid butterfly Bicyclus anynana (AGD98718) [50% amino acid (aa) identity and 67% similarity]Helicoverpa assulta (45% identity and 65% similarity)B. mori pgFAR2324S. littoralis  by RACE-PCR cDNA amplification and subsequent Sanger sequencing. These pgFARs encompass ORFs of 1,438 and 1,362 bp, corresponding to proteins of 476 and 454 aa, respectively. SlitpgFAR I and SexpgFAR I share 84% aa identity, SlitpgFAR II and SexpgFAR II share 93% aa identity, and SlitpgFAR I and SlitpgFAR II share 44% aa identity. A pair-wise sequence comparison of SexpgFAR I and SlitpgFAR I with other noctuid pgFARs (Heliothis and Helicoverpa spp.) showed more than 44% aa identity, whereas a comparison of SexpgFAR II and SlitpgFAR II with the same FARs (Heliothis and Helicoverpa spp.) showed 73% aa identity  based on their deduced amino acid sequences, which contain a conserved NAD(P)H-binding motif 2324identity .S. exigua moths revealed that both SexpgFAR I and SexpgFAR II were expressed only in females and were exclusively expressed in the PG  and B. mori (BmopgFAR) pgFARs, which are reported to be C16-acyl-specific FARs16Danaus plexippus (DplpgFAR) and Plutella xylostella (PxypgFAR) pgFARs  pgFARs , which a) pgFARs . In the AR clade .S. exigua pgFARs, we cloned the SexpgFAR I and SexpgFAR II ORFs into the pYES2.1 shuttle vector and transformed them into InvSc1 yeast cells. We also established InvSc1 yeast cells that were transformed with an empty vector (negative control) and B. mori pgFAR (positive control)14 acyl and C16 acyl compounds that naturally occur in yeast -11-hexdecenoic acid (Z11-16:acyl), one of the minor pheromone precursors, we performed a functional assay with Z11-16:acyl, and both pgFAR-transformed yeast constructs produced Z11-16:OH. However, SexpgFAR I produced significantly more Z11-16:OH than SexpgFAR II . The SexpgFAR II-transformed yeast cells produced minor amounts of Z11-16:OH , indicating reduced activity -14-hexdecenoic acid), an intermediate pheromone precursor of the Asian corn borer (ACB) O. furnacalis that is not found in the glands of Spodoptera species . SexpgFAR II was unable to convert E14-16:COOMe to the corresponding alcohol -11-tetradecenoic acid Me (E11-14:COOMe), (Z)-9-tetradecenoic acid Me (Z9-14:COOMe) and (Z)-11-tetradecenoic acid Me (Z11-14:COOMe) in separate experiments (single-substrate assay). Hexane extraction of the yeast pellets revealed large amounts of Z11-16:OH  and traces of E11-14:OH. None of the other substrates could be reduced to the corresponding alcohols . Similar functional assay experiments were performed with SlitpgFAR I and SlitpgFAR II, and these yielded similar results -12-tetradecenoic acid Me (E12-14:COOMe) and (Z)-12-tetradecenoic acid Me (Z12-14:COOMe) to the corresponding alcohols, and we observed significant Z12-14:OH production compared with all the other monounsaturated compounds , indicating the functional similarity of S. exigua pgFAR I and B. mori pgFAR. Both have evolved to reduce C16 acyl compounds.To further test the selectivity of pgFAR II, we performed an additional functional assay with odoptera . Interesompounds  and S4. ompounds . We repe14 fatty acid pheromone precursors -9,12-tetradecadienyl acetate (Z9E12-14:OAc), Z9-14:OAc, Z11-16:OAc and Z9-14:OH at a 34:40:4:22 ratioZ9-14:OH and 22% Z11-16:OH, but the yeast cells produced significantly less Z9E12-14:OH (16%) (Z11-16:OH with that observed in our previous single-substrate assay (see Z11-16:OH production by SexpgFAR II. Because we supplemented the yeast cells with higher quantities of Z11-16:COOMe (0.5 mM) in the single-substrate assays, SexpgFAR II, despite its low reductase activity to the Z11-16:acid, produced nearly half of the Z11-16:OH amount produced by SexpgFAR I  would reduce enzyme activity and decrease production of the di-unsaturated fatty alcohol Z9-14:OH production with a blend ratio of 73:27  . We compssay see  and founxpgFAR I . The rea alcohol . This exoduction , and we al assay . To furtof 73:27 . These eS. exigua pgFAR, we conducted two separate multi-substrate assay experiments, one with concentration ratios of Z9E12-14:COOMe to Z9E11-14:COOMe equal to 1:1 and another with concentration ratios of Z9E12-14:COOMe and E10E12-14:COOMe -9,11-tetradecadienyl acetate (Z9E11-14:OAc), Z9-14:OAc, E11-14:OAc, 14:OAc, Z11-14:OAc, and -10,12-tetradecadienyl acetate (E10E12-14:OAc) at a ratio of 57:11:11:1:7:14S. littoralis was recently characterized, but the reductase specificity for shaping the pheromone blend has not been demonstratedZ9E11-14:OH (28%), E11-14:OH (25%), Z11-14:OH (27%) and Z9-14:OH (20%) and a lower concentration of E10E12-14OH  was similar to that of E10E12-14:OH  and Z9E11-14:COOMe . S. littoralis pgFARII preferentially converted the Z9E12-14:acid to the corresponding alcohol  exclusively use the C12 and C14 mono-unsaturated fatty acetate compounds as a pheromone . We performed a functional assay with SexpgFAR II-transformed yeast cells supplemented with equal concentrations of five mono-unsaturated and di-unsaturated compounds  results for SexpgFAR II also showed that all of the monounsaturated methyl esters were converted to alcohols at approximately the same level; however 11-14:OH . We obta11-14:OH . This stSpodoptera spp. reported, S. frugiperda, S. pectinicornis and S. praefica use saturated and mono-unsaturated C12 acetate esters as pheromonesZ)-5-decenyl acid Me (Z5-10:COOMe) and (Z)-7-dodecenyl acid Me (Z7-12:COOMe). None of these compounds were converted into the corresponding alcohols (results not shown). The results indicate that SexpgFAR I and SexpgFAR II are unable to reduce any fatty acyl substrates shorter than C14.Among the 18 16 and C14 acyls, respectively, and demonstrate that both FARs are involved in the Spodoptera pheromone biosynthesis pathway to produce different precursor alcohol compounds. Our yeast functional assay data confirm that pgFAR I can primarily reduce C16 fatty acyl precursors, whereas pgFAR II was found to be involved in the reduction of a broad range of saturated and unsaturated C14 acyls. FAR could reduce neither C10 nor C12 fatty acyl precursors. The discovery of a C16-specific function of FAR I may not be consistent with the function of the previously identified FARs from corn borer mothsB. anynana FAR14, adding to the degree of specialization, as observed with the production of Z9E12-14:OH in S. exigua and Z9E11-14:OH in S. littoralis. The enzymatic activity and substrate specificity identified in this study clearly suggest that the Spodoptera female sex pheromone components are produced via de novo common biosynthetic routesS. exigua, found that SexpgFAR I and SexpgFAR II belong to the Lepidoptera-specific pgFAR lineage and demonstrated that these two genes encode specialized pheromone biosynthetic reductases. A recent study of the evolution of FAR genes in plants and animals clearly indicated that insects have undergone large expansions relative to the other taxa examined, which is consistent with a hypothesis that FAR is a multigene family that evolved through the birth-death evolution14S. exigua and S. littoralis, the presence of mono- and di-unsaturated C14 and C16 fatty acyl precursors might prime the expansion of two pgFARs in these species.We identified two active FARs that are exclusively expressed in the PG and exhibit specificity for CB. moriB. anynanaE11-14:OH by SexpgFAR I may result from an ancestral feature that is capable of reducing C14 and C16 acyl precursors. However, the ancestral activity was less specialized and reduced C14:acyl and E11-14:acyl. To confirm this hypothesis, we performed multi-substrate functional assays with BmopgFAR-transformed yeast cells supplemented with 10 different precursors as a positive control 38Z9E11-14:acid. In contrast to the reported PG extract ratio of E10E12-14:OAc (14%), we found significantly lower quantities of E10E12-14:OH (>4%)  . The expZ9-14:COOMe and Z9E12-14:COOMe resulted in higher Z9-14:OH production, indicating the selectivity of the S. exigua pgFAR system. We obtained pheromone blend ratios of the major pheromone compounds that were similar to those previously reportedSpodoptera pgFARs are selective for the Z9-14:acid, and S. exigua and S. littoralis exhibit a preference for its major precursors: Z9-14:acid, Z9E12-14:acid and Z9E11-14:acid, respectively. Of the 18 identified Spodoptera spp. pheromones, Z9-14:OAc is a major pheromone component, and 14 Spodoptera spp. use Z9E12-14:OAc in their pheromone blends  is a characteristic of this genus. Until now, no other Lepidoptera pgFARs have been reported to exhibit multifunctional activity toward mono- and di-unsaturated pheromone precursors71114151617The multi-substrate functional assay of SexpgFAR II with different blend ratios of e blends . Our funur study  and 6. AE10Z12-16:acid has been reported in B. moriE11-14:acid and Z11-14:acid, have been reported in O. nubilalisYponomeuta spp., Agrotis spp. Heliothis and Helicoverpa spp. reduce a broad range of C12, C14 and C16 acyl precursors but present clear specificity for the natural C14 pheromone precursors1517S. exigua and S. littoralis. pgFAR I in both species possesses similar functional activity, and its function might have evolved before radiation within the Spodoptera genus. We reiterate that the Spodoptera pgFAR I selectively recognizes the chain length of the substrates for pheromone biosynthesis, whereas pgFAR II accepts a broad range of substrates. pgFAR II is selective for the major pheromone precursors , which unequivocally supports the hypothesis that the S. exigua and S. littoralis pgFARs play important roles in shaping the final pheromone blend ratio. Overall, our findings demonstrate that two biosynthetic pgFARs are involved in Spodoptera. pgFAR II exhibits broad selectivity and acts on C14 mono- and di-unsaturated precursors, and pgFAR I exhibits narrow selectivity and acts on Z11-16:acyl to produce the moth pheromone signals. The mechanism through which Spodoptera pgFARs coordinate the reduction of the mono- and di-unsaturated C14 and C16 precursors remains to be elucidated. In most Spodoptera species, the precursor and pheromone component ratios exhibit significant disparities  dedicated to the reduction of C16:acid is functionally conserved in moth and butterfly lepidopteran lineages and that a multifunctional pgFAR II acts on the mono and di-unsaturated C14:acid precursors that are collectively involved in Spodoptera pheromone biosynthesis. Our study opens the door for a new area of research aiming to disclose the evolutionary changes associated with C14-specific and C16-specific FARs in moth pheromone biosynthesis.A single pgFAR with broad selectivity for C16 fatty acyl precursors with a distinct substrate specificity for parities , and thernis see . Our stun-hexane (250 ng/μL).The chemicals and authentic standard compounds mentioned in the supporting information (SI) were purchased from Pest Control of India Private Limited  and Pherobank (Netherlands). The compounds used for the functional assays were diluted in 95% ethanol (0.5 mM), and the standard compounds were diluted in Spodoptera exigua and S. littoralis pupae were originally collected from a vegetable garden , and laboratory cultures were maintained on an artificial dietS. exigua and S. littoralis are not endangered or protected species. The emerged females were separated daily prior to reaching scotophase, and their pheromone glands were dissectedS. exigua females using an RNeasy isolation kit (Qiagen). First-strand cDNAs were prepared using ArrayScript reverse transcriptase (Life Technologies). We created a dataset of FARs using EST, as previously reportedEscherichia coli JM109 cells (Promega). A total of 400 clones  was selected. The plasmid DNA was purified, and sequencing reactions were performed using a BigDye terminator kit v3.1 (Applied Biosystems) and subsequently analyzed on an ABI PRISM 3500 genetic analyzer (Life Technologies). The sequences were identified through BLASTx searches using the S. exigua FAR sequences as the queries. The 5′ and 3′ cDNA specimens were prepared with the SMART RACE amplification kit (Clontech) according to the manufacturer’s instructions. Gene-specific primers were designed (Table S2) and used in the PCR reactions to amplify the PCR products, which were gel-purified and ligated into the pGEM-T easy vector (Promega). The ligation products were transformed into Escherichia coli JM109 chemically competent cells (Promega). Plasmid DNA specimens were purified, sequenced, and analyzed using an ABI 3500 genetic analyzer. Separate sequencing reactions were established for primer walking to obtain the full-length pgFAR sequences, and the data were compiled using the BioEdit programWe extracted the total RNA from the pooled pheromone glands of 0- to 3-day-old virgin S. exigua moths (n = 3) and from the entire thorax and abdomen of male moths (n = 3) was prepared using an RNeasy mini kit (Qiagen). The first-strand cDNAs were synthesized from 1 μg of the total RNA using an ArrayScript RT kit (Life Technologies). Touchdown PCR was performed using 200 ng of the cDNA template and primer sets designed for the specific amplification of each FAR-like sequence  of 3-day-old female sequence . The conS. exigua moths at mid-scotophase. Total RNA was extracted (Qiagen), and cDNAs were synthesized using MultiScribe™ Reverse Transcriptase (Life Technologies). Three biological cDNA samples were prepared, and quadruplicate 25-μL quantitative PCR reactions for each transcript  were performed on an ABI 7500 instrument (Life Technologies). The template was 100 ng of PG cDNA, and SexpgFAR I, SexpgFAR II, and β-actin primers at a concentration of 10 μM were used (Table S2) (Tm = 60 °C). Premier Primer software  and the Power SYBR Green qPCR MasterMix were used (Life Technologies). The amplification efficiency (E) of each primer pair was initially assessed from serial dilutions of the cDNAs, and the raw cycle threshold values (Ct) were averaged from the quadruplicate reactions. The β-actin RNA was used for normalization, and all relative expression levels were calculated using the ABI 7500 program (Life Technologies). The fluorescence background baselines and amplification thresholds were calculated automatically. The fold changes in expression levels were calculated against a normalized (β-actin) sample and subjected to one-way analysis of variance (ANOVA). Means were compared using Tukey’s HSD (honestly significant difference) test in the SPSS programThree PGs were dissected from 2- to 3-day-old female S. exigua and S. littoralis FAR nucleotide sequences were used as queries for BLASTx searches in the GenBank database, and the nucleotide and amino acid sequences from different insect species were retrieved to construct a phylogenetic tree. Similarity analyses of the DNA and protein sequences were conducted, and multiple-sequence alignment was performed using the MUSCLE programThe S. exigua and S. littoralis pgFAR transcripts according to a previously described protocol7Saccharomyces cerevisiae  according to the protocol listed in the SI Materials and Methods. For each test sample in the functional assay, we included a negative control (vector only) and a positive control (B. mori pgFAR)B. mori pgFAR were purchased from Integrated DNA Technologies (Belgium).A yeast expression system (Invitrogen) was used to functionally express the B. mori pgFAR)77via ANOVA tests followed by Tukey’s test, and homogeneity-of-variance tests were performed using the SPSS program.The pgFAR yeast transformants were supplemented with different ratios of the precursors that were added at a total concentration of 0.5 mM. Single- and multi-substrate assays with different blend ratios (mentioned in the results) were performed using three replicates for each experiment as well as a negative control (vector only) and a positive control (Accession codes: The sequences reported in this paper have been deposited in the GenBank database (Accession Nos KF805977-KF805983 and KR781119-KR781121).How to cite this article: Antony, B. et al. Two fatty acyl reductases involved in moth pheromone biosynthesis. Sci. Rep.6, 29927; doi: 10.1038/srep29927 (2016).


Escherichia coli O serogroups (from O1 to O187), we developed a comprehensive and practical molecular O serogrouping (O genotyping) platform using a polymerase chain reaction (PCR) method, named E. coli O-genotyping PCR. Although, the validation assay using the PCR system showed that most of the tested strains were successfully classified into one of the O genotypes, it was impossible to classify 6.1% (35/575) of the strains, suggesting the presence of novel O genotypes. In this study, we conducted sequence analysis of O-AGCs from O-genotype untypeable Shiga toxin-producing E. coli (STEC) strains and identified six novel O genotypes; OgN1, OgN8, OgN9, OgN10, OgN12 and OgN31, with unique wzx and/or wzy O-antigen processing gene sequences. Additionally, to identify these novel O-genotypes, we designed specific PCR primers. A screen of O genotypes using O-genotype untypeable strains showed 13 STEC strains were classified into five novel O genotypes. The O genotyping at the molecular level of the O-AGC would aid in the characterization of E. coli isolates and will assist future studies in STEC epidemiology and phylogeny.Serotyping is one of the typing techniques used to classify strains within the same species. O-serogroup diversification shows a strong association with the genetic diversity of O-antigen biosynthesis genes. In a previous study, based on the O-antigen biosynthesis gene cluster (O-AGC) sequences of 184 known  Escherichia coli strains in taxonomical and epidemiological studies  in Denmark1, has recognized 185 E. coli O serogroups. These are designated O1 to O188 (publication of O182 to O188 is pending) and include three pairs of subgroups, O18ab/ac, O28ab/ac, and O112ab/ac; and six missing numbers, O31, O47, O67, O72, O93, and O122  and the histidine biosynthesis (his) operon. Sequence comparisons of O-antigen biosynthesis gene clusters (O-AGCs) indicate a variety of genetic structures (wzx/wzy and wzm/wzt) located on the O-AGCs are highly variable and can be used as gene markers for the identification of O serogroups via molecular approaches. So far, several studies have reported genetic methodologies allowing rapid and low-cost O-typing of isolates (E. coli O serogroups (from O1 to O187), and organized 162 DNA-based O serogroups (O-genotypes) on the basis of the wzx/wzy and wzm/wzt sequences. Subsequently we presented a comprehensive molecular O-typing scheme: an E. coli O-genotyping polymerase chain reaction (ECOG-PCR) system using 20 multiplex PCR sets containing 162 O-genotype-specific PCR primers  constitute one of the most important groups of food-borne pathogens, as they can cause gastroenteritis that may be complicated by hemorrhagic colitis or hemolytic-uremic syndrome  by the ECOG-PCR. By comparing sequences we revealed six novel O-genotypes and developed specific-PCRs for each novel O-genotype.E. coli O antigens . O genotypes were determined by ECOG-PCR as described in our previous study  and Shigella boydii type 13  single antisera were also used to test for the agglutination reaction.O serogroup were determined by agglutination tests in microtiter plates using commercially available pooled and single antisera against all recognized us study . SalmoneTable 1). All strains were isolated from human feces (including patients with diarrhea and hemorrhagic colitis) in Japan from 2008 to 2012. The O-AGC sequences flanked by wcaM and hisI were extracted from draft genome sequences determined using an Illumina MiSeq sequencer , as previously described (The O-AGC sequences were determined from six O-genotype untypeable (OgUT) STEC strains, of which four were serologically typeable  and two others were untypeable (OUT) strains  and their specificities were evaluated by using 185 O-serogroup reference strains (O1–O188) from SSI using the following PCR conditions. Genomic DNA from E. coli strains was purified using the Wizard Genomic DNA purification kit  or DNeasy Blood & Tissue Kit  according to the manufacturer’s instructions. PCRs were performed using 10 ng/μl of template DNA. PCR was performed as follows: each 30-μl reaction mixture contained 2 μl of genomic DNA, 6 μl of 5× Kapa Taq buffer, dNTP mix , MgCl2 , primers , and 0.8 U of Kapa Taq DNA polymerase . The thermocycling conditions were: 25 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 1 min. PCR products (2 μl) were electrophoresed in 1.5% agarose gels in 0.5× TBE , and photographed under UV light after the gel was stained with ethidium bromide (1 mg/ml).Polymerase chain reaction primers for specifically identifying novel O genotypes were designed . Four O-AGCs  were obtained from strains that were serologically classified into O1, O39, O40, and O141, respectively (Table 1). Two others (named OgN9 and OgN12 genotypes) were obtained from strains that were both serologically and genetically unclassified into any groups (Table 1). Actually, the OgN9 strain did not react with any particular antiserum, and OgN12 showed identical agglutination titers with O34 and O140 antisera, which resulted in OUT classsification. OgN8, OgN10, and OgN12 carried rmlBDAC for the synsthesis of deoxythymidine diphosphate (dTDP)-L-rhamnose, and OgN31 carried rmlBA-vioA for dTDP viosamine synthesis . OgN9 carried fnlA-qnlBC for UDP-N-acetyl-L-quinovosamine (UDP-L-QuiNAc) synthesis . All novel O-AGCs carried the wzx/wzy O-antigen processing genes . The wzx/wzy sequences from OgN O-AGCs were compared with those from 171 O-serogroup strains and 11 OX-group reference strains, indicating that their sequences were unique compared to those from known O-AGCs (less than 70% DNA sequence identity of closest pairs), except for wzx of OgN31 . The sequence of OgN31_wzx was 98.7% identical in DNA sequence (99.0% amino acid sequence identity) to that of O116. Sequence comparison of O-AGCs revealed that the left region including wzx and genes for the d-TDP glucose pathway was conserved between OgN31 and Og116, and the right region including the wzy and glycosyltransferase genes was unique (less than 40% DNA sequence identity) in each O-AGC . O-AGC gene sets from four pairs with members of different genotypes that agglutinated with the same O antisera were compared . Between OgN10 and Og1 (from O1 strain), and between OgN8 and Og141 (from O141 strain), rmlBDAC genes were highly conserved in both O-AGCs, while other genes including wzx and wzy were diversified (less than 70% DNA sequence identity). Between OgN31 and Og39 (from O39 strain), different types of sugar biosynthesis genes were located on each O-AGC . There was no genetic similarity between OgN1 and Og40 (from O40 strain). From these results, we were convinced that these six were novel O-AGCs.Six types of O-AGC were identified from OgUT STEC strains . OgN10 and OgN9 strains agglutinated with S. enterica O42 and S. boydii type 13 antisera, respectively (data not shown).A BLAST search of the NCBI database revealed that the OgN10 O-AGC is similar to that of Table 2 and Figure 4). All primer pairs were targeted unique sequences of wzy, except for OgN31 for which primers were targeted to a glycosyltransferase gene. Each PCR was evaluated by using all 185 O-serogroup reference strains from O1 to O188 and six novel O-genotype strains (listed in Table 1). PCR products of the expected sizes on the agarose gel were obtained only with the corresponding strains, and no extra products were observed in the size range between 100 and 1,500 bp (data not shown).Six PCR primer pairs were designed for identifying the novel O-genotypes . All 13 strains classified into five novel O-genotypes were eae-negative STEC, and OgN8, OgN10, and OgN31 had been isolated from patients with bloody diarrhea. OgN8 and OgN12 strains also cross-reacted with O41 and O34 antisera, respectively.Among 35 O-serogrouped E. coli from patients with urinary tract infections (E. coli (E. coli O1 strains could be generally subtyped into two genotypes, Og1 and OgN10, which were clearly linked to extra-intestinal/avian pathogenic E. coli and STEC, respectively. Among the O1 serogroup, three types of antigen structures have so far been reported (N-acetyl-D-mannosamine residue was suggested to be a common O1-specific epitope (E. coli. Another advantage for DNA-based typing is that serologically untypeable and ambiguous strains could be clearly classified. At the present time, the PCR-based method reported here is the only way to distinguish OgN9. Although, STEC OgN groups have not emerged as a major public health issue, these groups are believed to be a possible cause of diarrhea and bloody diarrhea. To gain more information about trends in STEC OgNs epidemiology, further studies of global OgN isolates are needed. The PCR method described in this study may help the surveillance and monitoring of the OgN groups. Additionally, published sequences from OgN O-AGCs may be used for other DNA-based methodologies, such as in silico typing using whole genome sequencing data (In this study, six novel O genotypes were revealed from STEC strains isolated from human patients, and the prevalence of these O genotype strains was confirmed in STECs. The OgN10 strains were serologically classified into the O1 serogroup. The O1-serogroup strain is often seen in extra-intestinal pathogenic fections  and septfections , and in (E. coli . Our pre(E. coli  and sequ(E. coli  and the (E. coli  carried (E. coli  were alsreported  and the  epitope . A partiing data .Conceived and designed the experiments: AI, MO, and TH. Performed the experiments: AI, SI, KS, and HN. Analyzed the data: AI and YO. Contributed reagents/materials/analysis tools: SI, KS, MO, and HM. Wrote the paper: AI. Critical revision of the paper for important intellectual content: AI, SI, and TH.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer BQ and handling Editor declared their shared affiliation and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


This study provides a new insight into the cross-talk between C/N and ABA signalling under the control of ABI1.ABI1 was identified as the corresponding gene of the C/N-nutrient response mutant  Arabidopsis FOX hunting population, which over-expresses full-length cDNAs for individuals. The resulting cni2-D (carbon/nitrogen insensitive 2-dominant) plant was found to overcome the post-germination growth checkpoint and to expand green cotyledons in disrupted high C/low N stress conditions. The CNI2 gene encodes ABI1, a phosphatase type 2C protein, which negatively regulates abscisic acid (ABA) signal transduction. Over-expressors of ABI1 were found to be insensitive to disrupted C/N stress, whereas the loss-of function mutant abi1-2 was hypersensitive, suggesting that ABI1 plays an essential role in the plant C/N response. By contrast, the C/N-dependent growth phenotype observed in wild-type plants was not associated with endogenous ABA content. Accordingly, the ABA-insensitive mutant abi1-1, which could not bind to the ABA–ABA receptor complex, was not insensitive and restored normal sensitivity to high C/low N stress. The canonical ABA signalling mutants abi4 and abi5 were also sensitive to disrupted C/N stress. Further gene expression analysis demonstrated that several genes in the SnRK2s and SnRK1s pathways are transcriptionally affected by high C/low N stress in wild-type plants regardless of the lack of increased endogenous ABA contents, whereas the expression of these genes were significantly suppressed in ABI1 over-expressors. Taken together, these results suggest direct cross-talk between C/N and non-canonical ABA signalling pathways, regulated by ABI1, in plants.Plants are able to sense and mediate the balance between carbon (C) and nitrogen (N) nutrient availability to optimize metabolism and growth, described as the C/N response. To clarify the C/N signalling mechanism, C/N-insensitive plants were obtained from an  Plant growth and development are controlled by many environmental factors and stresses, including nutrition, light, drought, and osmotic stress. Carbon and nitrogen are essential for plants, being constituents of nutrients and metabolites that provide energy and serve as constitutive molecular backbones. Moreover, these constitutive molecules also possess hormone-like functions, transducing signals to regulate plant growth and development (Arabidopsis FOX (Full-length cDNA Over-eXpressing) hunting system, which consists of independent transgenic lines expressing full-length cDNAs under the control of the CaMV promoter (carbon/nitrogen insensitive 1-D (cni1-D), and it was found that the CNI1 gene encoded the ubiquitin ligase ATL31  is critical for plant growth in response to environment challenges such as drought, salt, and osmotic stress. Additional exogenous ABA delayed germination and arrested plant growth, similar to plants exposed to excess sugar stress , with macni2-D (carbon/nitrogen insensitive 2-dominant) was isolated which was able to survive under extremely high C/low N stress conditions. The CNI2 gene encodes a type 2C protein phosphatase, ABI1, a negative regulator of ABA signalling. ABI1 is a central component of ABA signalling transduction, with its phosphatase activity inhibiting several SnRK2 proteins  and all other plant material used in this study were grown under the conditions described previously by Arabidopsis FOX hunting population was provided by RIKEN  and sequenced.The Surface-sterilized seeds were sown on MS medium modified with different concentrations of glucose and total nitrogen, as described by ABI1 cDNA fragment was amplified by PCR using the primers described in Supplementary Table S1 at JXB online. The fragment was sequenced and cloned into the pENTR/D-TOPO vector (Invitrogen) to generate the plasmid pENTR/ABI1. Full-length ABI1 cDNA was subsequently introduced into the pMDC83 T-DNA binary vector (ABI1 gene under the control of the 35S promoter (35S-ABI1). This 35S-ABI1 construct was used to transform Arabidopsis as described by A full-length y vector , accordiSupplementary Table S1 at JXB online. PCR products were electrophoresed on agarose gel and visualized by ethidium bromide staining. Quantitative RT-PCR (qRT-PCR) was performed using SYBR premix EX Taq (TAKARA) on an Mx3000P QPCR System (Agilent Technologies) according to the manufacturer’s protocol. The internal control for calculating ΔΔCt was 18S rRNA. The specific primer sets used for qRT-PCR analysis are shown in Supplementary Table S2 at JXB online.Total RNA was isolated from plants as described by Arabidopsis plantlets were analysed essentially as described by 6-ABA, Icon Isotopes, Summit, NJ, USA) at 4 °C for 1h. After clearing by centrifugation, the supernatant was evaporated and loaded onto an Oasis HLB column . The eluate containing ABA was evaporated and applied to an Oasis MCX column (Waters) to remove cationic compounds. After washing the column with 1% acetic acid, ABA was eluted with 80% acetonitrile containing 1% acetic acid. The eluate was evaporated and applied to an Osasis WAX column (Waters). After successive washes with 1% acetic acid and 80% acetonitrile, the acidic fraction containing ABA was eluted with 80% acetonitrile containing 1% acetic acid. This fraction was dried and dissolved in 1% acetic acid. ABA was determined by LC-MS/MS (Agilent 6410) using a ZORBAX Eclipse XDB-C18 column (Agilent).The ABA contents of carbon/nitrogen insensitive 2-D (cni2-D), which could continue post-germination growth in the extremely high C/low N medium . This protein has been shown negatively to regulate ABA signalling by inhibiting downstream SnRK2s kinase activities  and limited nitrogen (0.1mM N), termed high C/low N conditions. This screening resulted in the identification of a new C/N response mutant, N medium . Under tD plants .ABI1 gene was responsible for the cni2-D phenotype, transgenic Arabidopsis plants over-expressing ABI1 under the control of the CaMV 35S promoter (35S-ABI1) were grown in C/N stress medium. The transgenic nature of these plants was confirmed by genomic PCR and RT-PCR analyses  and high C/low N stress (200mM Glc/0.3mM N) media. Although WT plants showed growth defects in this C/N stress medium, 35S-ABI1 plants expanded green cotyledons and continued post-germination growth .To confirm that the analyses . C/N resRBCS1-B gene was lower in high C/low N stress conditions than in normal conditions, whereas expression of this gene in 35S-ABI1 plants was equal under both conditions  with various glucose concentrations  and at constant glucose concentration (200mM) with various nitrogen concentrations . Almost all WT, 35S-ABI1, and abi1-2 plants grew normally and expanded green-coloured cotyledons in 0mM Glc/0.3mM N medium .To evaluate the function of ABI1 in the plant C/N response further, growth of the N medium . The posN medium , C. The /0.3mM N , C, indi/0.3mM N , C. The 35S-ABI1, and abi1-2 plants could grow normally and expanded green cotyledons in 200mM Glc/3mM N medium, WT plant growth was relatively inhibited and showed a decreased greening ratio (30%) in 200mM Glc/1mM N. This inhibition was more apparent in the abi1-2 mutant (11%), while 35S-ABI1 plants were insensitive (78%)  , D. SeveArabidopsis seedlings and indicating that endogenous sugar levels positively affect ABA biosynthesis. However, the relationship between C/N availability and ABA biosynthesis has not been determined. To determine whether the C/N-induced growth defect is caused by an increased ABA level, endogenous ABA contents were measured in WT Arabidopsis seedlings grown under each C/N condition. WT seedlings were grown in medium containing different concentrations of glucose  and nitrogen  for 7 d  for 7 d were transferred to N-deficient medium (100mM Glc/0.3mM N). After 3 d, the ABA content of plants grown in N-deficient medium was slightly but significantly lower than that of plants in control conditions , similar limited . By conttransfer . These rabi1-1 mutant was examined. In abi1-1, the Gly180 residue is replaced by Asp, with the mutated ABI1 protein unable to bind to the ABA-receptor complex, resulting in constitutive inactivation of SnRK2 proteins  and abi5 (abi5-1) loss-of-function mutants were tested, since these genes encode key canonical transcriptional factors under the control of ABI1-SnRK2s (abi4 and abi5 mutants are not resistant to the extremely high C/low N stress condition (300mM G/0.1mM N) used in cni mutant screening  plants. The expression of CHS was about 13-fold higher in WT plants grown under high C/low N stress (200mM Glc/0.3mM N) than under control (100mM Glc/3mM N) conditions, whereas CHS induction was clearly suppressed in 35S-ABI1 plants (NCED3, which encodes an ABA-biosynthetic enzyme, was not increased by high C/low N stress in WT plants (NCED3 was slightly higher in 35S-ABI1 than in WT plants, suggesting that ABA content is not responsible for the insensitive phenotype of 35S-ABI1 plants under the high C/low N stress condition. Evaluation of the expression of several ABA-responsive marker genes in the SnRK2s pathway (RD29b expression in WT plants was about 6-fold higher under high C/low N stress than under control conditions, but that RD29b expression was slightly lower in 35S-ABI1 plants under stress conditions (LEA3-4 and TSPO genes (RAB18, AREB1, and ABF3 were not increased by high C/low N stress and were similar in WT and 35S-ABI1 plants (DIN6 and SEN5 (DIN6 and SEN5 in WT plants were significantly decreased in response to high C/low N stress, but were not affected in 35S-ABI1 plants (To assess the C/N-mediated signal transduction pathway under ABI1 control, transcript analysis was performed in WT and 1 plants , confirmT plants , a findiT plants . The expnditions . SimilarPO genes , E. By c1 plants –H. These1 plants , J. Takecni2-D plants isolated in this study were insensitive to disrupted C/N-nutrient stress conditions, with ABI1 being the gene responsible for the phenotype of these plants (ABI1 caused the successful post-germination growth of these plants under high C/low N stress condition, while the loss-of-function mutants showed hyper-sensitive responses and severe growth inhibition when compared with WT plants (Arabidopsis. ABI1 encodes a protein phosphatase and functions as an essential negative regulator of ABA signal transduction. It has been reported that ABA is involved in multiple stress signal mediations including the sugar and osmotic signals during post-germination growth. Sugar and osmotic stresses enhance ABA biosynthesis followed by inhibition of ABI1 and then activation of the ABA signalling cascade (abi1-1 mutant, in which mutated ABI1 is unable to bind to the ABA-receptor complex, resulting in the constitutive inactivation of SnRK2s, was not resistant to C/N stress (ABI4 and ABI5 were not insensitive to high C/low N stress conditions (The e plants . Over-exT plants , clearly cascade . This fiN stress . In addinditions , despiteNCED3, which encodes a key enzyme in ABA biosynthesis, under both high C/low N and limited-N conditions (It was found that ABA biosynthesis is affected by C/N status but is not associated with plant growth phenotype in response to C/N . ABA connditions , 6.Arabidopsis, transcription analysis showed that the expression of ABA biosynthetic genes could be activated by nitrogen starvation as well as by sugar supplementation (Arabidopsis plants grown at high and low nitrogen conditions (abi4 and abi5 and ABA-deficient mutants were shown to be less sensitive to the inhibitory effects of high nitrate medium on lateral root formation (Since little was known about the relationship between ABA biosynthesis and nitrogen availability, the results of this study are complicated. Related studies have reported that ABA biosynthesis in aerial parts of cucumbers is affected by limited nitrogen, but that this effect is dependent on time after treatment and developmental stage (RD29b, LEA3-4, and TSPO were increased by high C/low N stress, and suppressed in ABI1 over-expressing plants (RAB18, AREB1, and ABF3, were not affected by high C/low N stress, indicating that C/N signals are mediated by specific ABA signalling pathways. In addition, the involvement of the SnRK1s, a family of essential kinases associated with vast transcriptional events and metabolic reprogramming, restoring homeostasis, and inducing tolerance to energy starvation stress (DIN6 and SEN5, both of which are typical SnRK1s-responsive marker genes, in WT plants, but which effect was clearly suppressed in the ABI1 over-expressor (The results in this study raise the question about how ABI1 regulates post-germination growth in response to C/N. To explore the unknown signalling pathway mediated by C/N response under ABI1 control, transcript levels of several ABA-signalling marker genes regulated by the SnRK2s pathway were examined. Interestingly, although endogenous ABA contents were not up-regulated, the levels of expression of g plants , suggestxpressor . This fiJXB online.Supplementary data can be found at Supplementary Table S1. PCR Primers used for genotype and transcript analyses.Supplementary Table S2. Primers used for quantitative RT-PCR analyses.Supplementary Fig. S1. C/N response phenotype of ABI1 over-expressors.Supplementary Fig. S2. Osmotic stress response of abi1-2 mutants.


In vitro disease modeling based on induced pluripotent stem cells (iPSCs) provides a powerful system to study cellular pathophysiology, especially in combination with targeted genome editing and protocols to differentiate iPSCs into affected cell types. In this study, we established zinc-finger nuclease-mediated genome editing in primary fibroblasts and iPSCs generated from a mouse model for radiosensitive severe combined immunodeficiency (RS-SCID), a rare disorder characterized by cellular sensitivity to radiation and the absence of lymphocytes due to impaired DNA-dependent protein kinase (DNA-PK) activity. Our results demonstrate that gene editing in RS-SCID fibroblasts rescued DNA-PK dependent signaling to overcome radiosensitivity. Furthermore, in vitro T-cell differentiation from iPSCs was employed to model the stage-specific T-cell maturation block induced by the disease causing mutation. Genetic correction of the RS-SCID iPSCs restored T-lymphocyte maturation, polyclonal V(D)J recombination of the T-cell receptor followed by successful beta-selection. In conclusion, we provide proof that iPSC-based in vitro T-cell differentiation is a valuable paradigm for SCID disease modeling, which can be utilized to investigate disorders of T-cell development and to validate gene therapy strategies for T-cell deficiencies. Moreover, this study emphasizes the significance of designer nucleases as a tool for generating isogenic disease models and their future role in producing autologous, genetically corrected transplants for various clinical applications. in vitro disease modeling with induced pluripotent stem cells (iPSCs) offers a valuable complementation to in vivo studies. The goal of our study was to establish an in vitro disease model for severe combined immunodeficiency (SCID), a group of inherited disorders of the immune system characterized by the lack of T-lymphocytes. To this end, we generated iPSCs from fibroblasts of a radiosensitive SCID (RS-SCID) mouse model and established a protocol to recapitulate T-lymphopoiesis from iPSCs in vitro. We used designer nucleases to edit the underlying mutation in prkdc, the gene encoding DNA-PKcs, and demonstrated that genetic correction of the disease locus rescued DNA-PK dependent signaling, restored normal radiosensitivity, and enabled T-cell maturation and polyclonal T-cell receptor recombination. We hence provide proof that the combination of two promising technology platforms, iPSCs and designer nucleases, with a protocol to generate T-cells in vitro, represents a powerful paradigm for SCID disease modeling and the evaluation of therapeutic gene editing strategies. Furthermore, our system provides a basis for further development of iPSC-derived cell products with the potential for various clinical applications, including infusions of in vitro derived autologous T-cells to stabilize patients after hematopoietic stem cell transplantation.Due to the limited availability and lifespan of some primary cells,  In vitro disease modeling with induced pluripotent stem cells (iPSCs) provides a practical alternative, and the study of several disorders has benefitted enormously from the convergence of three key technologies: modern genomics that links genetic variants to disease phenotypes, the ability to generate patient-specific iPSCs that can be differentiated into cell types affected by disease, and powerful tools for editing complex genomes . Primary mouse ear fibroblasts were cultured in MEF medium . ES.CCE cells were cultivated in ES medium . iPSCs were cultivated in iPS medium  in a normoxic incubator on a shaker at 60 rpm. At day 2.5, 0.5 ml of EB medium plus cytokines rhBMP-4, activinA, rhVEGF165 and rhFGF-2 at 5 ng/ml final concentration each  was added. At day 8, EBs were harvested, washed with PBS and collected in Trypsin-EDTA, diluted 1:15 in Collagenase IV. After 30 min, 2.5 ml of cell dissociation buffer (Gibco) was added and cells transferred through a 70-μm mesh. Hematopoietic progenitor cells (HPCs) were washed with PBS and analyzed for CD41/cKit expression by flow cytometry prior to hematopoietic expansion. To this end, 106 EB-derived HPCs were cultivated for 3 days under hypoxic conditions in STFV medium . At day 3, cells were harvested through a 100-μm mesh and washed with PBS prior to in vitro T-cell differentiation. To this end, up to 3x105 expanded HPCs or 0.5-1x105 HSCs were added in T-cell differentiation medium . After 3 days, 2 ml medium was added and cultivation continued for up to 4 weeks. Every 7 days cells were harvested through a 100-μm mesh, washed with PBS, transferred to a new OP9-DL1 cell layer, and analyzed for T-cell differentiation by flow cytometry.The protocol was adapted from previously published work ,49. For For flow cytometric analysis, cells were resuspended in FACS buffer . To stain for pluripotency marker SSEA-1, iPSCs were rinsed with PBS and stained with biotinylated anti-SSEA-1 antibody (eBioscience) diluted in FACS buffer for 20 min at 4°C. After rinsing the secondary staining was performed with a streptavidin-APC antibody (eBioscience). Hematopoietic cells were pretreated with Mouse BD Fc block (BD Biosciences) before antibody staining. Antibody staining was performed for 20 min at 4°C. EB-derived HPCs were stained with CD41-PE, cKit-APC, or respective isotype controls . iPSC-derived T-cells were stained with CD44-PE and CD25-APC, or CD4-PE and CD8-APC. Viability staining with 7-AAD was performed for 2 min during the last rinsing, before samples were measured on a FACSCalibur. Alternatively, iPSC-derived T-cells were stained with CD45-APC-Cy7, CD4-PerCPR-Cy5.5, CD8-PE-Cy7 , CD44-PE, CD25-APC, TCRß-FITC (eBioscience) and DAPI, before analysis on a FACSCanto II with FACSDiva (BD Biosciences). All samples were analyzed with FlowJo software (Tree Star). T-cell receptor diversity was analyzed by CDR3 spectratyping as previously described .t-test with unequal variance.All experiments were performed at least three times. Error bars represent standard deviation (SD). Statistical significance was determined with a two-sided Student's http://www.ncbi.nlm.nih.gov/nuccore) accession number for the ZFN target site in intron 84 of the prkdc gene on mouse chromosome 16 is AB030754: 189732.The National Center for Biotechnology Information (NCBI) Nucleotide database (S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S1 Figprkdc. The position and sequence of the ZFN BS in intron 84 is shown. F1, F2, F3 indicate target triplets for each binding half-sites. The spacer is highlighted in italics, mut* indicates the position of the SCID mutation. (B) Sequence of prkdc-specific ZF modules. Amino acid sequences of the ZF modules that recognize F1, F2, or F3 target triplets for the left and right target half-sites (5’ to 3’ orientation). Three ZFs for the left binding half-site  and two for the right target half-site  have been selected and tested for their ability to activate a beta-galactosidase  reporter . (C) Expression analysis of ZFNs. ZFN-encoding plasmids were transfected in 293T cells and protein levels detected by immunoblotting using Odyssey IRDye antibodies. L1, L2, L3, ZFN left subunits 1, 2 or 3 with WT FokI domain; L1H, L2H, L3H, ZFN left subunits 1, 2 or 3 with “EA” obligate heterodimeric FokI domain ; L2N, ZFN left subunit L2 with “EA” obligate heterodimeric FokI domain without NLS signal; R1, R2, ZFN right subunits 1 or 2 with WT FokI domain; R1H, R2H, ZFN right subunits 1 or 2 with “QK” obligate heterodimeric FokI domain ; R1N, R2N, ZFN right subunits 1 or 2 with “QK” obligate heterodimeric FokI domain without NLS signal. GFP served as transfection and loading control. (D) In vitro cleavage assay. ZFN pair L2N and R1N was in vitro transcribed/translated and mixed with a ZFN BS-containing PCR product. Cleavage reaction  was analyzed on a 1.5% agarose gel. Size markers are indicated. (E) Plasmid-based gene correction assay. 293T cells were transfected with target plasmid, repair matrix and ZFN or SceI plasmids in order to induce episomal homology-directed repair . Y axis shows the percentage of relative gene correction frequency, which is calculated as GFP-positive cells (target plasmid corrected) REX-positive cells (transfected cells). (+), positive control SceI; (-), negative control SceI∆; WT, L2 + R1 with homodimeric FokI domain; OH, L2 + R1 with obligate heterodimeric FokI domain;-NLS, L2 + R1 with OH FokI domain without NLS signal.(A) Schematic view of ZFN binding site (ZFN BS) in (TIF)Click here for additional data file.S2 FigA) Ssea-1 expression. Surface expression of pluripotency marker Ssea-1 was measured by flow cytometry. Fib.S, NOD.SCID-derived ear fibroblasts; ES.CCE, murine ES cell line; iPS.S1, iPS.S6 and iPS.S12, NOD.SCID-derived iPSC clones. (B) Alkaline phosphatase staining. NOD.SCID-derived iPSCs were stained using the Alkaline Phosphatase Detection Kit and analyzed by microscopy. (C) Spectral karyotyping (SKY). Multicolor fluorescent in situ hybridization (FISH) based karyotyping was used to assess genome integrity. iPS.T8 and iPS.T44, gene targeted iPSC clones. (D) Teratoma Assay. Hematoxylin/eosin-stained sections of teratoma isolated 8 weeks after injection of iPSCs into NSG mice. Detection of ectodermal, endodermal and mesodermal tissues. iPS.T8 and iPS.T44, gene targeted iPSC clones.((TIF)Click here for additional data file.S3 FigA) Excision of the reprogramming cassette. iPSCs have been treated with Flp recombinase to remove the lentiviral reprogramming cassette. Excision was confirmed by detection of the viral PRE elements via Southern blot. Genomic DNA was digested with EcoRV or BamHI. Positions of restriction sites in reprogramming cassette and the expected minimal band sizes are indicated on top. M, DNA size marker; S6 and S6X, SCID iPSC clones; T25 and T25X, targeted iPSC clones; WT and WTX, wild-type iPSC clones; X indicates clones with excised reprogramming cassette. (B) Random integration of donor. The NeoR cassette, as an indicator of donor DNA, was detected by Southern blot to confirm targeted integration in prkdc intron 84. Genomic DNA was digested with EcoRV or NdeI. Positions of restriction sites in the modified intron 84 and the expected band sizes are indicated on top. (C) Determination of vector copy number (VCN). Copy number of the lentiviral reprogramming vector was assessed by quantitative PCR [Click here for additional data file.S4 FigSamples were stained with CD41-PE, cKit-APC or their corresponding isotype controls, and 7-AAD after dissociation of 8 d matured embryoid bodies (EBs). All plots were pre-gated on FSC/SSC and 7-AAD-negativity. Red numbers indicate percentage of cells in each quadrant. iPS.WT and iPS.WT X, wild-type iPSC clones; iPS.S6 and iPS.S6 X, SCID iPSC clones; iPS.T25, iPS.T25X and iPS.T44, targeted iPSC clones; X indicates iPSC clones with excised reprogramming cassette.(TIF)Click here for additional data file.S5 Figin vitro generated T cells. Exemplarily shown are PCR analyses of the variable beta chains Vß1, Vß6, Vß8.1, Vß8.3, Vß10, Vß12, Vß14 and Vß20. X axis indicates PCR fragment size in bp, Y axis shows quantity of PCR amplicons. Thymus, control DNA of cells isolated from thymus; HSC, in vitro generated T cells; iPS.WTX, wild-type iPSC clone; iPS.S6 and iPS.S6X, SCID iPSC clones; iPS.T25, iPS.T25X and iPS.T44, targeted iPSC clones; X indicates clones with excised reprogramming cassette.Quantitative PCR was performed on genomic DNA isolated from (TIF)Click here for additional data file.


Despite recent efforts in discovering novel long non-coding RNAs (lncRNAs) and unveiling their functions in a wide range of biological processes their applications as biotechnological or therapeutic tools are still at their infancy. We have recently shown that AS Uchl1, a natural lncRNA antisense to the Parkinson's disease-associated gene Ubiquitin carboxyl-terminal esterase L1 (Uchl1), is able to increase UchL1 protein synthesis at post-transcriptional level. Its activity requires two RNA elements: an embedded inverted SINEB2 sequence to increase translation and the overlapping region to target its sense mRNA. This functional organization is shared with several mouse lncRNAs antisense to protein coding genes. The potential use of AS Uchl1-derived lncRNAs as enhancers of target mRNA translation remains unexplored. Here we define AS Uchl1 as the representative member of a new functional class of natural and synthetic antisense lncRNAs that activate translation. We named this class of RNAs SINEUPs for their requirement of the inverted SINEB2 sequence to UP-regulate translation in a gene-specific manner. The overlapping region is indicated as the Binding Doman (BD) while the embedded inverted SINEB2 element is the Effector Domain (ED). By swapping BD, synthetic SINEUPs are designed targeting mRNAs of interest. SINEUPs function in an array of cell lines and can be efficiently directed toward N-terminally tagged proteins. Their biological activity is retained in a miniaturized version within the range of small RNAs length. Its modular structure was exploited to successfully design synthetic SINEUPs targeting endogenous Parkinson's disease-associated DJ-1 and proved to be active in different neuronal cell lines. In summary, SINEUPs represent the first scalable tool to increase synthesis of proteins of interest. We propose SINEUPs as reagents for molecular biology experiments, in protein manufacturing as well as in therapy of haploinsufficiencies. Large genomic projects such as ENCODE .The nervous system appears as a privileged site for lncRNA expression, as the vast majority of these transcripts is brain-enriched and regulates neuronal development and functions  and siRNAs as tools to decrease gene expression of pathological target genes such as, for example, mutant huntingtin in Huntington's disease -associated genes, we recently discovered AS Uchl1, a natural lncRNA antisense to Ubiquitin carboxyl-terminal esterase L1 (Uchl1/PARK5)  to UP-regulate translation of partially overlapping sense mRNAs. The overlapping region is indicated as the Binding Doman (BD) while the embedded inverted SINEB2 element is the Effector Domain (ED). By swapping BD, synthetic SINEUPs are designed targeting mRNAs of interest.in vivo representing a new type of RNA-based drug for molecular therapy.This work shows that synthetic SINEUPs are a versatile tool to increase synthesis of target proteins of interest paving the way for future applications of SINEUPs as molecular biology reagents and for manufacturing of recombinant proteins. Most importantly, SINEUPs may be directed to selected mRNA species Plasmids expressing target proteins were previously described. In particular, for this study we used pEGFP-C2  has been described in Carrieri et al. . SINEUP FWD AS 2xFLAG:ATATCTCGAGAATTCCTTGTCATCGTCGTCCTTGTAGTCCATCAATTCCAGCACACTGGCGGCCGTREV AS 2xFLAG:GAGAGATATCCTCGGATCCACTAGTAACGGCCGCCAGTGTGCTGGAATTGATGGACTACAAGGACGPrimers were annealed, elongated by PCR, digested and ligated into XhoI-EcoRV sites of SINEUP-backbone.Short SINEUP targeting GFP (miniSINEUP-GFP) was generated combining BD of SINEUP-GFP and ED of AS Uchl1. Briefly, inverted SINEB2 was PCR amplified and cloned into EcoRI and HindIII sites of pcDNA3.1(-). SINEUP-GFP BD was subsequently added to the inverted SINEB2-containing plasmid at XhoI and EcoRI sites to obtain miniSINEUP-GFP. The following primers were used:FWD EcoRI InvSINEB2: TATAGAATTCCAGTGCTAGAGGAGGREV HindIII InvSINEB2: GAGAAAGCTTAAGAGACTGGAGCFWD ApaI all O/L: TATAGGGCCCTCTAGACTCGAGREV EcoRI O/L GFP20: GAGAGAATTCCAGCACAGTGGCGGCCGCSINEUPs targeting DJ-1 were generated by annealing and PCR-based method (−40/+32) or with annealing and ligation of phosphorylated oligonucleotides (−40/+4), using the following primers:SINEUP-DJ-1 (−40/+32) FWD:ATATCTCGAGGCCAGGATGACCAGAGCTCTTTTGGAAGCCATTTTTATGTTATATGTTTSINEUP-DJ-1 (−40/+32) REV:GAGAGATATCTTTCAGCCTGGTGTGGGGCTTGTAAACATATAACATAAAAATGGCTTSINEUP-DJ-1 (−40/+4) FWD:TCGAGCCATTTTTATGTTATATGTTTACAAGCCCCACACCAGGCTGAAASINEUP-DJ-1 (−40/+4) REV:TTTCAGCCTGGTGTGGGGCTTGTAAACATATAACATAAAAATGGCAll constructs were verified by sequencing.HEK 293T/17 cells were obtained from ATCC (Cat. No. ATCC-CRL-11268 293T/17) and maintained in culture with Dulbecco's Modified Eagle Medium (GIBCO) supplemented with 10% FBS (SIGMA) and 1% antibiotics (penicillin/streptomycin), as suggested by the vendor. HepG2 cells were kindly provided by Professor Collavin L. from the University of Trieste, Italy  at 1 μM for 1 h or at 100 nM for 16 h. DNA damage was induced by doxorubicin (SIGMA), at 1 μM for 1 h or 500 nM for 16 h.HEK 293T/17 cells were transfected with Fugene HD (Roche) and Lipofectamine 2000 (Life Technologies), following manufacture's instruction. HepG2, HeLa, SH-SY5Y, BE(2)M17 and SK-N-SH cells were transfected with Lipofectamine 2000. All cells were transfected with a 1:6 ratio between sense and SINEUP encoding plasmids, maintaining the conditions described for S/AS Uchl1  following manufacture's instructions.Data of RNA and protein levels were obtained from the same transfection in each replica.For Western blot analysis, cell pellets were directly resuspended in Laemli sample buffer, briefly sonicated, boiled and loaded on poly-acrilamide gels.Primary antibodies used in this study include anti-GFP rabbit polyclonal antibody , used 1:1000, anti-FLAG M2 , 1:1000, anti-β-actin (SIGMA), 1:5000, and anti-TRAF6 (Abnova), used 1:500. To detect endogenous DJ-1 protein an antibody produced in our laboratory was used  following manufacturer's instructions. RNA was treated with on-column DNase I (QIAGEN) followed by a second DNase I digestion in solution (Ambion). Two rounds of DNase digestion were required to avoid plasmid DNA contamination in this experimental setting. Single strand cDNA was prepared from 1 μg of purified RNA using the iSCRIPT™ cDNA Synthesis Kit (Bio-Rad) according to manufacturer's instructions. qRT-PCR reaction was performed on diluted cDNA (1:20) using SYBR-Green PCR Master Mix (Applied Biosystem) and an iCycler IQ Real time PCR System (Bio-Rad). Relative expression was calculated with the ΔΔCt method  .in vitro are routinely used as model systems to study the molecular mechanisms of gene functions as well as cell factories to produce therapeutic proteins. Considering the flexibility of SINEUP technology and its potential applications in molecular biology experiments, protein manufacturing and therapeutics, we investigated the efficacy and reproducibility of SINEUPs in different cell lines in vitro. To this purpose we selected hepatocellular carcinoma HepG2 cells, for their use as a model system by large multicenter consortia, epithelial carcinoma HeLa cells for their wide use in cell biology and in therapeutic protein production as well as HEK 293T/17 cells as positive control of SINEUP activity. As a representative synthetic SINEUP, we took advantage of SINEUP-GFP to increase GFP protein levels in transient overexpression experiments.Mammalian cell cultures p > 0.05).We estimated SINEUP activity as fold changes in protein levels encoded by targeted mRNAs in the presence/absence of SINEUP with mRNA amounts kept constant (Similar fold-changes were observed for SINEUP activity with GFP target at 24 and 48 h after transfection (data not shown). However timing for optimal activity was cell-line dependent, as best conditions were found at 24 h in HeLa and 48 h in HEK 293T/17 and HepG2 cells Figures .p > 0.05), albeit HEK 293T/17 cells tended to be more effective  and 2.3 with Fugene HD (n = 6 experiments) (data not shown). Under these experimental conditions, RNA from transfected SINEUP-GFP was detected in the cytoplasmic fraction although a prominent accumulation in the nucleus was evident  was designed to mimic the molecular anatomy of SINEUP-GFP. In particular, SINEUP-FLAG BD encompasses −40 nucleotides (in pcDNA3 plasmid backbone) before FLAG-initiating Met and +32 bases covering the first FLAG tag sequence Figure .p = 0.46). Interestingly, increased TRAF6 levels could be measured when probing lysates with anti-TRAF6 specific antibody  .A 50% up-regulation was measured with FLAG-DJ-1 and 90% with FLAG-Hba. In both cases, sense mRNA in -SINEUP and +SINEUP transfections was not statistically different  long with BD of 72 base pairs, ED of 170 base pairs in addition to intervening sequences, a partial Alu element (73 base pairs) and a 3′ tail. This length is suitable for delivery systems such as viral vectors, but incompatible with the use of SINEUPs as naked RNA therapeutic molecules. Therefore, we aimed at synthesizing the shortest functional SINEUP that retains its translation enhancement activity. MiniSINEUP-GFP was obtained combining SINEUP-GFP BD and ED from AS Uchl1   Figure . Interes) Figure . Under tThe use of SINEUPs as a versatile tool to increase protein synthesis is strictly dependent on their ability to act on endogenous, cellular mRNAs transcribed from a genomic locus that does not present a natural SINEUP antisense gene.in vitro system to study the function of PD-associated genes. Cells transfected with an empty vector were used as control (-SINEUP).To prove this crucial point we designed synthetic SINEUPs targeted to endogenous DJ-1 mRNA, a gene involved in recessive familial Parkinson's Disease (PD). We generated two SINEUP-DJ-1 constructs with two different BD elements: −40/+32 , from −40 nucleotides (in annotated 5′ untranslated region) before DJ-1 translation start site to +32 bases in the coding sequence, as well as −40/+4 , ending at the first nucleotide at 3′ of ATG Figure . TogetheAs shown in Figure In summary synthetic SINEUPs can be designed to mRNAs of interest that do not present a natural SINEUP in their genomic locus opening up the scalable use of SINEUPs to target endogenous protein coding transcripts of mammalian cells.To our knowledge SINEUPs are the only tool available so far that uses lncRNAs to enhance translation of target proteins. Their modular structure allows synthetic design of an overlapping region (BD) to target proteins of interest, without changing the overall structure of the original lncRNA. Indeed, by extracting solely the target-selecting BD and the ED from the original lncRNA, SINEUPs can be resized to miniSINEUPs retaining their activity.Over competing technologies, SINEUPs have two major advantages: (1) they modulate translation of target mRNAs without introducing stable genomic changes into target cells; (2) their induction of selected protein is typically in a more physiological range (2-fold) than most conventional gene replacement strategies. These features render SINEUPs (and miniSINEUPs) a potentially interesting tool for a number of applications. First, SINEUPs may be used as reagents for molecular biology. As siRNAs have become an invaluable instrument to inhibit gene expression, many cases exist in which increasing the amount of a specific protein is required. SINEUPs may be designed to a single gene of interest or to a tag that is common to several targets and achieve translation enhancement, thus formally becoming the opposite counterpart of siRNAs. This is especially relevant in the nervous system where regulation of translation has an enormous impact on synaptic plasticity and memory formation. The synthesis of specific proteins requires a fine tuned control at single synapse and spines involving the translation of selective subtypes of mRNAs. The special challenges posed by the anatomo-functional organization of the brain require cellular machinery for controlling mRNAs localization according to the morphology and connectivity of single neuronal cell types. This is achieved at least in part by the use of different TSSs leading to the synthesis of mRNA isoforms with specific 5′UTR containing information for subcellular localization and translation  to identify the correct AUG-surrounding region of the mRNA of interest. Importantly, we observed that high number of cell passages negatively influence SINEUP activity (data not shown). Global CAP-dependent translation is maintained through mTOR activity . Stefano Gustincich, Piero Carninci and Silvia Zucchelli are named inventors in patent issued in the US Patent and Trademark Office on SINEUPs and licensed to TransSINE Technologies. Michael H. Jones is CEO of Cell Guidance Systems, a company distributing SINEUPs as laboratory reagents. Stefano Gustincich and Piero Carninci are co-founders of PARKscreen, an Italian SME aimed to use and develop therapeutic SINEUPs.Stefano Gustincich, Piero Carninci, Claudio Santoro, Michael H. Jones and Silvia Zucchelli declare competing financial interests as co-founders and members of TransSINE Technologies (


NR1H3 gene associated with the susceptibility to systemic lupus erythematosus (SLE).Liver X receptors are established sensors of lipid and cholesterol homeostasis. Recent studies have reported that these receptors are involved in the regulation of inflammation and immune responses. We attempted to identify single nucleotide polymorphisms (SNPs) of the NR1H3 gene promoter polymorphisms were analyzed using a luciferase assay, real-time polymerase chain reaction, B cell proliferation assay and an electrophoretic mobility shift assay.SNPs were genotyped using SNaPSHOT assay in 300 Korean patients with SLE and 217 normal controls (NC), and in replication samples (160 SLE patients and 143 NC). Also, the functional effects of P = 0.009, P = 0.034 and P <0.001, respectively). Proliferation of the -1830 TC type was increased compared to the -1830 TT type in basal, GW3965 and T0901317 treated B cells from SLE patients . Transcription factor GATA-3 preferentially bound the -1830 T allele in the promoter.We identified five polymorphisms: -1851 T > C (rs3758673), -1830 T > C (rs3758674), -1003 G > A (new), -840 C > A (rs61896015) and -115 G > A (rs12221497). There was a significant and reproducible difference in the -1830 T > C, -1003 G > A and -115 G > A polymorphisms between the SLE and the NC. Luciferase activity of the structure containing -1830 C was less enhanced compared to the structure containing -1830 T in basal, GW3965 and T0901317 treated Hep3B cells (NR1H3 genetic polymorphisms may be associated with disease susceptibility and clinical manifestations of SLE. Specifically, -1830 T > C polymorphism within NR1H3 promoter region may be involved in regulation of NR1H3 expression. Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by dysregulation of the immune system involving the hyperactivity of T and B cells, elevated production of pathogenic autoantibodies, complement activation, and the formation of immune complexes causing multiorgan damage by deposition in host tissue. AlthougNR1H3) and beta (NR1H2) can influence macrophage biology by modulation of lipid metabolism and by effects on innate immunity. The release of cytokines including interleukin (IL)-1, IL-6 and tumor necrosis factor-α from macrophages results in recruitment of monocytes and cross-talk with T cells, perpetuates cellular activation and further promotes atherosclerotic lesion developmentpropyloxy]phenylacetic acid hydrochloride  or 5 μmol/L N--N-phenyl]-benzenesulfonamide  for 24 hours. These concentrations and time were chosen based on previous workCAAAGGCTCT - 3′, CdxA as a nonspecific competitor, 5′ - AGATCTGGTACCATTTAAGCCCTCGAGATCTA - 3′ and for GATA-3 as a specific competitor, 5′ - GTTATTTATCTCTTAGTTGTAGTTATTTATCTCTTGTTGTA - 3′. The double-stranded oligonucleotides were annealed by incubation at 95°C for five minutes followed by gradual cooling to room temperature. The double-stranded DNA probes were radiolabeled at their 5′-ends with DNA polymerase I  and [α-32P]CTP. The reaction mixtures (10 μl), which contained 5 μg of nuclear extract, 10 mM HEPES (pH 7.9), 250 ng poly(dI:dC), 0.005 mM MgCl2, 75 mM NaCl, 5 mM DTT, 1 mM NaNO3, 10 mM NaF, 1 mM EDTA, 400 ng bovine serum albumin and 50% glycerol, were preincubated on ice for 20 minutes, and then incubated for 20 minutes on ice in the presence of 0.2 pmol of radiolabeled probe. The reaction mixtures were then subjected to electrophoresis on a native 6% polyacrylamide gel. For competition experiments, unlabeled blunt-ended competitor oligonucleotides were added to the binding reaction mixtures before the addition of the radiolabeled oligonucleotide probes. The gels were dried and radioactivity was detected using a FLA3000 scanner .Hep3B cells were washed with ice-cold phosphate-buffered saline before being suspended in ice-cold hypoosmotic buffer comprised of 10 mM HEPES/KOH (pH 7.9), 2 mM MgCl2, 0.1 mM ethylenediaminetetraactic acid (EDTA), 10 mM KCl, 1 mM dithiothreitol (DTT), 1 mM phenylmethanesulfonylfluoride (PMSF), 1 mM NaVO3, 10 mM NaF and complete protease inhibitor cocktail . The cells were incubated on ice for 10 minutes and for an additional 15 minutes in the presence of 0.2% Nonidet P-40. After centrifugation at 15,000 × g for 30 seconds, the pellet was resuspended in a cold saline buffer that contained 50 Mm HEPES/KOH (pH 7.9), 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 10% (v/v) glycerol, 1 mM DTT, 1 mM PMSF and protease inhibitors. The mixture was incubated on ice for 30 minutes. After centrifugation at 15,000 × g for 30 minutes at 4°C, the supernatant containing the nuclear proteins was stored at -80°C until use. The following double-stranded oligonucleotides were used as probes for the electrophoretic mobility shift assay (EMSA): for P values <0.05 were considered to be significant. Haplotypes were analyzed using Haploview version 4.2 based on the EM algorithm  (Table Linkage disequilibrium between SNPs and locus by locus was examined. Two genetic polymorphisms of the NR1H3 gene are summarized in Table P = 0.005). Also, arthritis (P = 0.006), anti-dsDNA (P = 0.041) and triglyceride (P = 0.011) were more common in the SLE patients with the -1003 A allele than the -1003 G allele, but oral ulcer (P = 0.039) and central nervous system (CNS) involvement (P = 0.015) were less common in the -1003 A allele. In addition, arthritis (P <0.001) was more common in the SLE patients with the -115 A allele than -115 C allele, but oral ulcer (P = 0.024) was less common in the -115 A allele ; however, arthritis was more common in patients with HT3 [TCGG] . The frequency of lymphopenia was significantly lower in patients who had haplotype HT2 [CTGG]       was analyzed in Hep3B cells and COS-7 cells. The luciferase activity of -1830 C was less enhanced when compared to that of -1830 T in GW3965 and T0901317 treated Hep3B cells         between the promoter region and the second intron region. Also, we found that the rare alleles of the -1830 T > C, -1003 G > A and -115 G > A polymorphisms were associated with a significantly higher disease susceptibility. In the haplotype analysis, there was a significant difference between SLE and NC in the observed haplotype HT1 [TTGG] and HT3 [TCAA]. Our results suggest that the human NR1H3 gene plays an important role in the development of SLE.In the present study, we evaluated the associations of genetic polymorphisms of the LXR genes have been reported in different ethnic populations and diseases, such as obesity; however, arthritis was more common in patients with HT3 [TCGG]. The frequency of lymphopenia was significantly lower in patients who had haplotype HT2 [CTGG]. These findings suggest that the disease phenotype is more common in patients with SLE who have minor allele -1830 T > C, -1003 G > A and -115 G > A polymorphisms than in those who have the major homozygous genotype. These phenotype results were consistent with the genotype results.In particular, the -1830 T > C promoter polymorphism was significantly different in genotype analysis and clinical manifestations. The -1830 T > C polymorphisms were located in the promoter site, which is important for gene expression. Therefore, we focused on the functional effects of -1830 T > C polymorphism. To determine if the genetic variants created a transcription factor binding site, sequences were submitted to the TFSEARCH online program, which revealed that the -1830 T > C polymorphism might be a potential GATA-3 binding motif. GATA-3 belongs to the GATA family of transcription factors. The GATNR1H3 because a shifted band was found in the presence of the -1830 T probe in the EMSA results. The transcription factor bound to the -1830 T probe was identified as GATA-3 through a competition assay, because GATA-3 was bound to the -1830 T allele promoter with a higher affinity compared with that of the 1830 C allele promoter. We also performed supershift assays using a commercially available anti-GATA-3 antibody and found that the anti-GATA-3 antibody could affect DNA-protein complex formation in the -1830 T probe. The -1830 T specific band was also competed by anti-GATA-3 antibody. Our data suggest that, in the transcription regulation of NR1H3 gene, GATA-3 might act as a positive regulator because the -1830 T allele containing the reporter construct showed higher promoter activity than the -1830 C allele containing the reporter construct. The results of the present study indicate that the binding affinity of GATA-3 may be decreased in SLE patients who carry the -1830 C allele.We performed an EMSA with nuclear extracts from Hep3B cells using double-stranded oligonucleotide probes that corresponded to the -1830 T > C. This polymorphism affected a transcription factor binding in the promoter region of The B cell proliferation assay was performed to determine the effect of -1830 T > C polymorphism. Proliferation of -1830 TC type was increased when compared to that of -1830 TT type in basal, GW3965- and T0901317-treated B cells from SLE patients. Also, proliferation of the -1830 TC type was increased when compared to the -1830 TT type in T0901317-treated B cells from NC. LXR agonist treated cells proliferated less than untreated cells. These findings are consistent with the results of previous studies that have shown that LXR agonist suppresses cell proliferation.NR1H3 mRNA expression level than the -1830 TT type B cells in SLE. Also, the -1830 TC type B cells displayed a lower NR1H3 mRNA expression level than the -1830 TT type B cells in NC. These mRNA expression and proliferation assay results show that the LXR reduced proliferation of B cells from SLE patients.Moreover, -1830 TC type B cells displayed a lower In our luciferase assay and proliferation assay, LXR was activated by both T0901317 and GW3965 that has a similar tendency but the T0901317 effect was stronger than that of GW3965. These findings are consistent with the results of previous studies that have shown that activation of PXR targets may explain why T0901317 induces dramatic liver steatosis, while GW3965 has a milder effect.NR1H3 gene genetic polymorphisms may be associated with disease susceptibility and clinical manifestations of SLE. Especially, the -1830 T > C polymorphism within the NR1H3 promoter region may be involved in regulation of NR1H3 expression.These results suggest that the ACR: American College of Rheumatology; bp: base pair; CI: confidence interval; CNS: central nervous system; co: co-dominant; (D)MEM: (Dulbecco’s) modified Eagle’s medium; do: dominant; DTT: dithiothreitol; EBV: Epstein-Barr virus; EDTA: ethylenediaminetetraactic acid; EMSA: electrophoretic mobility shift assay; FBS: fetal bovine serum; HWE: Hardy-Weinberg equilibrium; Ig: immunoglobulin; IL: interleukin; LPS: lipopolysaccharide; LXR: liver X receptor; MAF: minor allele frequency; NA: not applicable; NC: normal control; OR: odds ratio; PCR: polymerase chain reaction; PMSF: phenylmethanesulfonylfluoride; re: recessive; RT-PCR: reverse transcription PCR; SLE: systemic lupus erythematosus; SNP: single nucleotide polymorphism.The authors declare that they have no competing interests.JYJ: conception and design, data collection and analysis, manuscript writing and final approval of the manuscript. JYN: data collection and analysis, critical revision and final approval of the manuscript. HAK: data collection and analysis, critical revision and final approval of the manuscript. YBP: data collection and analysis, critical revision and final approval of the manuscript. SCB: data collection and analysis, critical revision and final approval of the manuscript. CHS: conception and design, data collection and analysis, manuscript writing and final approval of the manuscript. All authors read and approved the final manuscript. Clinical characteristics of the study subjects.Click here for fileComparison of the clinical characteristics according to the genotype of NR1H3 gene in SLE.Click here for fileComparison of the clinical characteristics according to the haplotype of NR1H3 gene in SLE.Click here for file


Corynebacterium ulceribovis strain IMMIB L-1395T (= DSM 45146T) is an aerobic to facultative anaerobic, Gram-positive, non-spore-forming, non-motile rod-shaped bacterium that was isolated from the skin of the udder of a cow, in Schleswig Holstein, Germany. The cell wall of C. ulceribovis contains corynemycolic acids. The cellular fatty acids are those described for the genus Corynebacterium, but tuberculostearic acid is not present. Here we describe the features of C. ulceribovis strain IMMIB L-1395T, together with genome sequence information and its annotation. The 2,300,451 bp long genome containing 2,104 protein-coding genes and 54 RNA-encoding genes and is part of the Genomic Encyclopedia of Type Strains, Phase I: the one thousand microbial genomes (KMG) project.The online version of this article (doi:10.1186/s40793-015-0036-7) contains supplementary material, which is available to authorized users. Corynebacterium ulceribovis IMMIB L-1395T (= DSM 45146 = CCUG 55727) was first isolated from the skin of the udder of a cow with a profound ulceration  , aconitase [EC:4.2.1.3] , isocitrate dehydrogenase [EC:1.1.1.42] , α-ketoglutarate dehydrogenase [EC:2.3.1.61] , succinyl-CoA:acetate CoA-transferase [EC:3.8.3.18] , succinate dehydrogenase [EC:1.3.5.1] , fumarate hydatase [EC:4.2.1.2] , NAD-dependent malate dehydrogenase [EC:1.1.1.37]  and FAD-dependent malate:quinone oxidoreductase [EC:1.1.5.4] . The gene encoding for the anaplerotic enzyme phosphoenolpyruvate carboxylase , which catalyzes the synthesis of oxaloacetate from pyruvate was also identified. Genes coding for the enzymes of the anaplerotic glyoxylate cycle, isocitrate lyase  and malate synthase , were present in the genome.A set of genes encoding enzymes necessary to drive a complete oxidative tricarboxylic acid cycle were found in the genome of C. ulceribovis was equipped with the genes encoding proteins envolved in glycogen biosynthesis by the classical GlgC/GlgA and the GlgE pathways. Key genes encoding enzymes involved in the GlgC/GlgA pathway include: glgC, encoding for glucose-1-phosphate adenyltransferase GlgC  which catalyzes the production of ADP-glucose from ATP and glucose-1-phosphate; glgA, encoding for glycogen synthase GlcA  which catalyzes the successive addition of glucose from the glycosyl-nucleotide to the growing α-1,4-linked chain to generate the linear glucan; and glgB, encoding for the α-1,4-glucan branching enzyme GlgB  which catalyzes the formation of α-1,6-glucosidic linkages in the linear α-1,4-glucans to produce glycogen.Glycogen, a soluble α-linked glucose polymer (or α-glucan) with ~90 % α-1,4-links in its backbone and ~10 % α-1,6-linked branches, is a source of carbon and energy storage in a wide variety of organisms, including bacteria . InspecttreS, pep2, glgE and glgB) encoding for the enzymes involved in this pathway were found: the glgE gene encoding for maltosyl transferase GlgE , the pep2 gene encoding a maltokinase Pep2  and the previously mentioned glgB gene encoding for the GlgB . BLASTP analysis revealed that the Pep2 protein is a maltokinase which forms a complex with trehalose synthase TreS. This is not surprising partly due to the fact that the pep2  gene is usually linked with the treS gene and in some micro-organisms like Psuedomonas entamophila, Rubrobacter xylanophilus and in numerous members of the class Actinobacteria the two genes are fused into a single gene  A3ECDRAFT_0568), which catalyzes the conversion of meso-DAP to L-lysine, is also present in the genome.apeptide , is an i-pathway , 92. TheC. ulceribovis genome harbors seven genes encoding for penicillin binding proteins (PBPs) that catalyze the final steps of peptidoglycan synthesis. Two genes encoding for the bi-functional high molecular-mass (HMM) PBP of 1A family, designated PBP1A (A3ECDRAFT_2000) and PBP1B (A3ECDRAFT_0034), which have transpeptidase-transglycosylase activities and catalyze the elongation of the uncross-linked glycan chains of the PG. Three genes encoding for HMM PBP 2 family, designated PBP2A (A3ECDRAFT_2052), PBP2B [FstL] (A3ECDRAFT_1562) and PBP2C (A3ECDRAFT_0722), which contain transpeptidase domains and an additional dimerisation domains and believed to play a role in cell morphogenesis. Two genes encoding for low-molecular mass (LMM) PBP of type PBP4, designated DacB (A3ECDRAFT_1828), and one of type PBP 5/6, designated DacC . The Dac proteins are D,D-carboxypeptidases which catalyze the cleavage of the terminal D-Ala-D-Ala bond making the stem peptide unavailable for transpeptidation and through this activity regulate the amount of cross-linking that can occur within the PG , which is reduced by dihydrofolate reductase [EC:1.5.1.3] to tetrahydrofolate.Genes encoding for all the enzymes of the folate biosynthetic pathways are present  via pantothenic acid from aspartate and α-ketoisovalerate , a gene (A3ECDRAFT_1818) encoded for a predicted oxidoreductase, which contains short-chain dehydrogenase (SDR) and DUF2520 domains, was present in the genome. BLASTP analysis revealed that the protein (A3ECDRAFT_1818) was 41 % identical to ketopantoate reductase (PanE/ApbA) in Corynebacterium durum F0235. Homologs of this KPR protein are present in other bacterisa such as Enterococcus faecalis V583 (EF1861), Francisellanovicidia (FTT1388) and Clostridium difficile. The KPR protein has been shown to also catalyze the conversion of 2-dehydropantoate to pantoate in Francisella species  which subsequently converted to pABA by aminodeoxychorismate lyase [EC:4.1.3.38]. The enzyme dihydropteroate synthase also catalyzes the condensation of pABA with 6-hydroxymethyl 7,8-dihydropterin pyrophosphate yielding dihydropteroate.In the C. ulceribovis DSM 45146T. The thiamine biosynthetic pathway of C. ulceribovis 45146T is outlined in  is derived from an oxidative condensation of cysteine, glyciene and 1-deoxy-D-xylulose 5-phosphate (DXP). Several genes such as thiG, thiO, iscS, and Dxs are involved in this process.Thiamine in its active form, thiamine pyrophosphate, is an essential cofactor for several microbial enzymes of the carbohydrate metabolism . The genC. ulceribovis 45146T carries the genes encoding for enzymes involved in NAD biosynthesis via both the canonical de novo pathway from L-aspartate and the salvage biosynthetic pathway from nicotinamide. In the de novo pathway, nicotinic acid mononucleotide (NaMN) is synthesized in three enzymatic steps from L-aspartate followed by two enzymatic steps to complete the synthesis of NAD  -BioM (A3ECDRAFT_0763) –BioN (A3ECDRAFT_0762), which constitute tripartite biotin transporter . The consesus sequences of the direct repeats of the two CRISPR regions are identical having a length of 28 bp (GTGTTCCCCGCGCAGGCGGGGATGAGCC) and separated by spacers with variable nucleotide sequences. CRISPRs provide the cell with aquired immunity to protect against bacteriophages, plasmids and other mobile genetic elements by a RNA interference-like mechanism [Moreover, echanism , 114.Secreted proteins play essential roles in bacteria, including the colonization of niches and host-pathogen interactions , 116. InC. ulceribovis DSM 45146T genome revealed the presence of all genes encoding proteins for the Sec translocation system. These include proteins forming the main membrane channel-forming complex SecYEG (A3ECDRAFT_0227/A3ECDRAFT_0157/A3ECDRAFT_0912), the cytosolic ATPase SecA (A3ECDRAFT_0372 and A3ECDRAFT_1078), the auxiliary proteins SecD (A3ECDRAFT_0848), SecF (A3ECDRAFT_0849) and YajC (A3ECDRAFT_0847), and the chaperones Ffh (A3ECDRAFT_0690) and FtsY (A3ECDRAFT_0689). As in other Gram-positive bacteria, the genome lacks homologs of the SecB protein, the chaperone that targets protein to the Sec translocon for passage through the cytoplasmic membranes. Genes encoding for the twin-arginine translocase (Tat) system, tatA/E, tatB, tatC, and tatD were also present in the genome. Like the majority of other sequenced actinobacterial genomes, the tatA/E gene (A3ECDRAFT_0977) was found next to tatC (A3ECDRAFT_0978), while the tatB gene (A3ECDRAFT_0538) and the tatD gene (A3ECDRAFT_1228) were separately located. The distinguishing feature of the TAT system is its ability to translocate fully folded proteins across the cytoplasmic membrane using the transmembrane proton gradient as the main driving force for translocation [Inspection of location .tad (tight adherence) locus in Haemophilus actinomycetemcomitans, was identified in the genome of C. ulceribovis. The genes of this tad locus appear to be organized as two adjacent clusters. The first cluster contained four genes encoding for: homolog of the TadZ protein (A3ECDRAFT_0049), followed by the TadA protein (A3ECDRAFT_0050), followed by two integral membrane proteins, TadB (A3ECDRAFT_0051) and TadC (A3ECDRAFT_0052). The second cluster contained three genes encoding for: a low-molecular weight protein (68 aa) containing DUF4244 domain (A3ECDRAFT_0053), followed by an unkown protein (A3ECDRAFT_0054), followed by a TadE-like protein (A3ECDRAFT_0055). Not linked to the tad locus, a gene encoding a putative prepilin peptidase PilD (A3ECDRAFT_0873), which was found located distantly in the genome. The tad export apparatus facilitates the export and assembly of pili, which mediate the nonspecific adhesion of bacteria to surfaces and are essential for host colonization and pathogenesis [A putative type IVb pilus-encoding gene cluster, similar to the ogenesis –120.C. ulceribovis DSM 45146T genome. A region, comparable to region 4 (ESX-4) of M. tuberculosis H37Rv, containig nine genes (A3ECDRAFT_0240- A3ECDRAFT_0248) was identified  and CFP-10 (product of esxB) are members of a protein family that is characterized by a length of ~100 amino acid residues, containing a WXG motif and lack a distinguishable Sec-signal sequence [M. tuberculosis and Staphylococcus aureus [In addition, genes encoding proteins for type VII secretion system ESX/T7SS) were also present in SS were asequence . Both ofs aureus , 123. Tys aureus , 125.FigC. ulceribovis provided crucial insights into the broad biological functions of this organism. Genome analysis showed that the overall features of C. ulceribovis are similar to those of the genus Corynebacterium; it possesses a complete set of peptidoglycan biosynthesis genes, synthesizes meso-DAP from aspartate via the dehydrogenase pathway, possesses all genes for menaquinone biosynthesis from corismate and has complete set of genes for the biosynthesis and processing of mycolic acids. C. ulceribovis also possesses a single fas1 gene encoding type I fatty acid synthase FAS I for de novo fatty acids biosynthesis and a complete set of genes associated with fatty acid degradation by the β-oxidation pathway. Genes encoding enzymes associated with the central carbohydrates metabolism were identified. C. ulceribovis possesses a complete TCA cycle and glyoxylate shunt; a functional PPP for generation of pentoses and NADPH for anabolic purposes; all gene necessary for glycogen metabolism; trehalose synthesis via the OtsA-OtsB pathway. The genome also contains genes encoding myo-inositol- 3-phosphate synthase and inositol monophosphatase involved in the biosynthesis of myo-inositol from glucose-6-phosphate as well as gene encoding for α-mannosyltransferase PimA leading to the synthesis of PIM. To meet cofactor requirements, several genes encoding for enzymes that catalyze de novo biosynthetic pathways for several cofactors are present in the genome. Finally the genome of C. ulceribovis harbors genes encoding proteins that protect the cells against the danger of bacteriophage infections. These include type I restriction enzymes (R-M enzymes), Abi-like protein that mediate bacteriophage resistance by abortive infection (Abi system) and CRISPER/cas system that serve as molecular “vaccination cards”.The availability of high-quality genome sequence from