The more than 120 genotypes of human enteroviruses (HEVs) reflect a wide range of evolutionary divergence, and there are 23 currently classified as human enterovirus C species (HEV-C). Two new HEV-C (EV-C117 and EV-C118) were identified in the Community-Acquired Pneumonia Pediatric Research Initiative (CAP-PRI) study, and the present paper describes the characterisation of the complete genome of one EV-C117 strain (LIT22) and two EV-C118 (ISR38 and ISR10) strains. The EV-C117 and EV-C118 5′UTR sequences were related to those of EV-C104, EV-C105 and EV-C109, and were slightly shorter than those of other HEV A-D species. Similarity plot analyses showed that EV-C117 and EV-C118 have a P1 region that is highly divergent from that of the other HEV-C, and phylogenetic analyses highly supported a monophyletic group consisting of EV-C117, EV-C118, EV-C104, EV-C105 and EV-C109 strains. Phylogenetic, Simplot and Bootscan analyses indicated that recombination was not the main mechanism of EV-C117 and EV-C118 evolution, thus strengthening the hypothesis of the monophyletic origin of the coding regions, as in the case of other HEV-C. Phylogenetic analysis also revealed the emergence of a new group within HEV-C that is divided into two subgroups. Nucleotide and amino acid identity in VP1 sequences have been established as useful criteria for assigning new HEV types, but analysis of the complete P1 region improves resolution. Picornaviridae family and cause a wide range of clinical conditions, ranging from asymptomatic or mild infections to more severe diseases such as acute hemorrhagic conjunctivitis, aseptic meningitis, severe community-acquired pneumonia (CAP), gastroenteritis, acute flaccid paralysis, myocarditis, and neonatal sepsis-like disease Human enteroviruses (HEVs) belong to the www.picornaviridae.com/enterovirus/hev-c/hev-c.htm), a number of which have been identified in the last decade. EV-C104 and EV-C109, which were detected in patients with respiratory syndrome in Switzerland and Nicaragua Enterovirus typing is based on comparing the sequences encoding the VP1 capsid protein: viruses of different genotypes have <75% nucleotide identity, and <85% amino acid identity The aims of this study were: i) to characterise the complete genome of one EV-C117 (LIT22) strain and two EV-C118 strains ; and ii) to investigate the relationship between these new viruses and other HEV-C strains.The study was approved by the Ethics Committee of the participating Centers: Università degli Studi di Milano, Milan, Italy; The Pediatric Infectious Disease Unit, Soroka University Medical Center, Beer-Sheva, Israel; Vilnius University Clinic of Children's Diseases, Vilnius, Lithuania; Fondazione IRCCS Policlinico San Matteo, Pavia, Italy. It was conducted in accordance with the standards of Good Clinical Practice according to the principles of the Declaration of Helsinki; the children's parents or legal guardians gave their written informed consent before the children were enrolled.The CAP-PRI study was a prospective community-based study designed to evaluate the prevalence of respiratory virus infections in children admitted to seven pediatric hospitals in Italy, Israel, Greece, Portugal, Lithuania and Romania because of radiographically confirmed CAP. Study enrolment began on 1 November 2010 and ended on 31 March 2011. A nasopharyngeal sample was collected from all of the children upon admission using a flexible pernasal flocked swab that was immediately placed in a mini-tube containing 1 mL of universal transport medium . The samples were stored at 4°C in the hospital laboratory until they were sent in a refrigerated package to the central laboratory , where they were aliquoted and stored at −80°C.Picornaviridae Study Group (www.picornastudygroup.com), compared with other enterovirus sequences, and named as proposed new enterovirus types EV-C117 (accession number JQ446368) and EV-C118 (JQ768163).Viral nucleic acids were extracted from the nasopharyngeal swabs by means of a Nuclisens EasyMAG automated extraction system , and the extracts were tested for respiratory viruses using the RVP Fast assay in accordance with the manufacturer's instructions . The samples that were positive for enterovirus/rhinovirus were retested in order to identify the rhinovirus. Phylogenetic analyses of the VP4/VP2 region showed that some nucleotide sequences belonged to the HEV species. The complete VP1 of two of these samples was submitted to the 2, and 2 µL of c-DNA template.We analysed one EV-C117 strain (LIT22) and two EV-C118 strains (ISR38 and ISR10). The complete genome sequence was obtained using degenerate primers designed by means of the multiple alignment of the EV-C104 and EV-C109 genomes available in GenBank, and additional primers designed on the basis of the first and subsequent rounds of sequencing in accordance with the primer walking method (the primer sequences are available upon request). Briefly, the eluted RNA was transcribed into cDNA using Moloney's murine reverse trascriptase  and random hexamers for one hour at 37°C. The MMLV-RT was subsequently denatured at 70°C, and the PCR was carried out in a final volume of 50 µL containing the virus-specific oligonucleotide primers (0.2 µM each), 2 U AmpliTaq Gold 360 DNA polymerase , 1× reaction buffer, 0.2 mM of each dNTP, 2 mM MgClThe PCR products were gel-purified using the Wizard SV Gel and PCR Clean-Up System , and then sequenced in both directions using the same forward and reverse primers as those used in the PCR. The nucleotide sequences were obtained by means of automated DNA sequencing using an ABI PRISM 3730 genetic analyser (Applied Biosystems). The 5′ and 3′ segment sequences were determined using the 5′–3′ rapid amplification of cDNA ends (RACE) in accordance with the manufacturer's instructions , and the sequences were assembled using Sequencher software, version 4.6 .http://www.cbs.dtu.dk/services/NetPicoRNA), in order to predict the picornaviral protease cleavage sites . The RNA secondary structure was predicted using the RNAalifold The sequences were aligned using the ClustalW program integrated in the MEGA package, version 5.0 The complete genome sequences of EV-C117 strain LIT22, EV-C118 strain ISR10 and EV-C118 strain ISR38 were deposited in GenBank and received the accession numbers JX232682, JX961708, and JX961709, respectively. These sequences were compared with 21 complete genomes of other HEV-C strains: EV-C104 strain AK11 (AB686524); EV-C104 strain CL-12310945 (EU840733); EV-C109 isolate NICA08-4327 (GQ865517); EV-C104 strain Pav262-11228 (JX982259); EV-C105 (JX514943); EV-C116 (JX514942); CV-A19 strain 8663 (AF499641); CV-A22 strain Chulman (AF499643); CV-A1 strain Tompkins (AF499635); EV-C96 strain BAN00-10488 (EF015886); EV-C99 (EF555644); CV-A21 strain Kuykendall (AF546702); CV-A24 (D90457); CV-A13 strain Flores (AF499637); CV-A11 strain Belgium-1 (AF499636); CV-A17 strain G12 (AF499639); EV-C102 (EF555645); CV-A20 strain IH35(AF499642); poliovirus type 3 strain Leon (K01392); poliovirus type 1 strain Mahoney (V01148); and poliovirus type 2 strain Sabin (X00595).The genome lengths of the EV-C117 and the two EV-C118 strains were respectively 7,362 nt and 7357 nt. Both genomes had a typical enterovirus organisation, with a coding region of 2,206 codons translated into four structural  and seven non-structural proteins . The genome of EV-C118 strain ISR10 differed from that of EV-C118 strain ISR38 in 57 nucleotide positions (0.77% divergence) scattered in the 5′UTR, and the P1, P2 and P3 regions. A single amino acid change (0.04% divergence) was observed in the P2 region.cis-acting replication element (CRE) was detected in the region of protein 2C (2CX8CX4C) in the 2C protein, the RNA-binding domain KFRDI in the 3C protein, and the two catalytic triads of H20-D38-C109 in the 2A protein and H40-E71-C147 in the 3C protein. The 3D NTP-binding motif (S/T)KVEQGKS was observed with one change in the EV-C117 genome (KVEAGKS), and with three changes in the EV-C118 genome (SKIKAGKS).The 5′UTR consisted of 666 nt in the EV-C117 genome and 671 nt in the EV-C118 genome. The phylogenetic tree constructed on the basis of the 5′UTR sequences showed that EV-C117 and EV-C118 clustered in a group that was different from that of the other HEV-C prototype strains , and conotein 2C , and hadSimilarity plot analysis showed that the P1 regions of EV-C117 and EV-C118 were highly divergent from those of the other HEV-C . In the As shown in Separate maximum-likelihood nucleotide sequence phylogenies were constructed for the complete genome, and the P1, P2 and P3 regions of EV-C117, EV-C118 and the other HEV-C . As showIn the P1 region tree, the position of the EV-C117 strain was partially unresolved and there was a low bootstrap value for specific bipartition , whereasThe nucleotide sequence identity of EV-C117 and EV-C118 with the other HEV-C types was plotted against their amino acid sequence identity . As showIn order to resolve the intra- and inter-group variability further, we analysed the nucleotide and amino acid pairwise distance within groups IIIa and IIIb, and in relation to the other HEV-C groups , by geneOver the last ten years, a number of new HEVs have been described as being responsible for respiratory syndromes, including EV-68, EV-C104, EV-C105, EV-C109, and EV-C116. Most of these HEV strains belong to HEV-C species cis-acting replication element (CRE) in the form of a small RNA hairpin in the coding region of protein 2C, which has been shown to be critical for RNA replication in picornaviruses Both EV-C117 and EV-C118 have the typical enterovirus genome organisation Intra- and intertypical recombinations are common within HEV-C et al. to define a new HEV type (75% VP1 nucleotide identity and 85 or 88% VP1 amino acid identity) VP1 is the most exposed capsid protein and is usually more variable than other virus genes. The VP1 sequences of EV-C117 and EV-C118 fulfilled the molecular typing criteria proposed by Oberste The phylogenetic trees constructed using the sequences of the P1, P2 and P3 regions were comparable with those constructed using the sequences of the entire genome. EV-C117 was closely related to EV-C104, whereas EV-C118 grouped with EV-C105 and EV-C109 in all of the trees (excluding P1). The HEV-C fell into three different groups, with group III consisting entirely of HEV strains with shorter 5′UTR sequences that have been identified during the last five years, including EV-C117 and EV-C118. As the recent detection of a number of HEVs belonging to this new group may be the result of increased surveillance and improved molecular methods, additional isolates that evolved along lineages that are independent of the original reference strain are needed in order to clarify the relationships within the group. Finally, phylogenetic analyses showed that group III is divided into two subgroups (IIIa and IIIb) with high bootstrap support, although the 5′UTR sequences from the two subgroups clustered together. These findings suggest the stringent conservation of a 5′UTR containing a secondary structure essential for virus replication, or the silent circulation of these viruses followed by viral evolution due to the accumulation of point mutations in the coding region.In conclusion, our findings characterise the complete genome of two new HEV-C. Analysis of the variation of the EV-C117 and EV-C118 sequences within the enterovirus species revealed greater sequence homology in the 5′UTR, less homology in the P1 region, and greater homology in the P2 and P3 region. The phylogenetic analysis showed the emergence of a new group within HEV-C, divided into two subgroups. Nucleotide and amino acid identity in VP1 sequences have been established as useful criteria for assigning new HEV types, but analysis of the complete P1 region improves the resolution of their identification and characterisation.


Despite the low level of structural order, the recombinant DAPk-DD retained the ability to interact with ERK2 in a 1∶1 ratio with a Kd in the low micromolar range. Only the full-length DAPk-DD could bind ERK2, indicating that the apparent ‘D-motif’ located in the putative sixth helix of DAPk-DD is not sufficient for ERK2 recognition. CD analysis revealed that binding of DAPk-DD to ERK2 is not accompanied by a significant change in secondary structure. Taken together our data argue that the DAPk-DD, when expressed in isolation, does not adopt a classical DD fold, yet in this state retains the capacity to interact with at least one of its binding partners. The lack of a stable globular structure for the DAPk-DD may reflect either that its folding would be supported by interactions absent in our experimental set-up, or a limitation in the structural bioinformatics assignment of the three-dimensional structure.Death-associated protein kinase (DAPk) is a calcium/calmodulin-regulated Ser/Thr-protein kinase that functions at an important point of integration for cell death signaling pathways. DAPk has a structurally unique multi-domain architecture, including a C-terminally positioned death domain (DD) that is a positive regulator of DAPk activity. In this study, recombinant DAPk-DD was observed to aggregate readily and could not be prepared in sufficient yield for structural analysis. However, DAPk-DD could be obtained as a soluble protein in the form of a translational fusion protein with the B1 domain of streptococcal protein G. In contrast to other DDs that adopt the canonical six amphipathic α-helices arranged in a compact fold, the DAPk-DD was found to possess surprisingly low regular secondary structure content and an absence of a stable globular fold, as determined by circular dichroism (CD), NMR spectroscopy and a temperature-dependent fluorescence assay. Furthermore, we measured the  The expression of the gene encoding death-associated protein kinase  was originally identified as being essential for the execution of apoptosis induced by IFN−γ The death fold superfamily of proteins includes the following four families: death domains (DDs), death effector domains (DEDs), caspase recruitment domains (CARDs) and PYRIN domains (PYDs). Whilst these protein families can be distinguished on the basis of pairwise sequence similarities, structure determination of representative members of each family has revealed that they share a common architecture consisting of six amphipathic α-helices (H1−H6) arranged in a compact, mostly antiparallel topology in which the N- and C-terminii are close together. Within each of the death fold families, the buried residues of H2 through H5 exhibit relatively high sequence similarity, whereas H1 and H6 are more highly divergent. The primary function of the death fold superfamily of protein modules is to mediate protein-protein interaction by forming predominantly homotypic associations, thereby underpinning the formation of multi-subunit signaling complexes. The extensive amino acid sequence diversity of surface-exposed residues in the death folds provides a wide range of physicochemical properties. Nevertheless most if not all contacts observed in DD complexes appear to fall into one of three topologically similar arrangements denoted: (i) type I, negatively charged residues of H2 and H3 of one DD interact with positively charged residues of H1 and H4 of another DD; (ii) type II, H4 and the H4–H5 loop of one DD interact with the N-terminal end of H6 of another DD; and (iii) type III, H3 of one DD interacts with the H1–H2 and H3–H4 loops of a second DD in vivoin vivo and in vitroSecondary structure prediction and sequence analysis for the DAPk-DD region suggests the presence of the expected six α-helices. Moreover hydrophobic residues are appropriately placed to align with key residues whose side chains are known to constitute the core of multiple examples of DD 3D structures . ParticuE. coli, purified and subjected to analytical gel filtration, circular dichroism (CD), NMR spectroscopy and a temperature-dependent fluorescence assay. The DAPk-DD was found to be a monomer at low micromolar concentrations that has surprisingly low regular secondary structure content, which has not been seen previously for other members of the DD family. Furthermore, we probed the interaction between ERK2 and various recombinant DAPk-DD constructs through in vitro binding and isothermal titration calorimetry (ITC) experiments. Despite the observed low level of structural order, the recombinant DAPk-DD retains the ability to interact with ERK2 in a 1∶1 ratio with a Kd in the low micromolar range. Taken together our data argue that the DAPk-DD, when expressed in isolation, is not stable as a classical 3D DD fold, yet in this state retains the capacity to interact with at minimum one of its reported binding partners.In order to further elucidate its role in the regulation of the DAPk function we set out to investigate the three-dimensional structure of the DAPk-DD. Recombinant DAPk-DD was over-expressed as a soluble fusion protein in 5′-GATGATGAT GGATCCTCA CAGGCCAGC CTCGG-3′) and reverse (5′-ATCATCATC CTCGAGCAC AGAGGATGC CTTCAGC-3′) primers, and inserted between the BamHI and XhoI sites in the plasmid GEV2, which encodes an N-terminal appendage of the B1 immunoglobulin binding domain of streptococcal protein G (GB1), a well-known solubilization enhancement tag 6-tag. The corresponding expressed protein product is denoted GB1-DAPk-DD(S).DAPk-DD expression constructs were prepared from the full-length human DAPk cDNA inserted into the pcDNA3 vector, kindly provided by Prof. A. Kimchi . The DAPk-DD DNA sequence (residues 1293−1399) was amplified using the forward  with forward (5′-GTGTTCAAA ATCAACCTG GATGGCAAT GGCCTCGAG CACCACCAC CACCACCAC TGAGATCC-3′) and reverse 5′-GGATCTCAG TGGTGGTGGT GGTGGTGCT CGAGGCCAT TGCCATCCA GGTTGATTT TGAACAC-3′) primers. Preparation of the truncated GB1-DAPk-DD constructs used for ERK-2 binding experiments was carried out in a similar fashion. All of these constructs also included a C-terminal His6-tag and a thrombin recognition sequence between the GB1 and DAPk-DD domains.A second, longer DAPK-DD expression construct, GB1-DAPk-DD(L), comprising residues 1293−1408 was prepared by mutagenesis of a separately prepared GEV expression vector that includes the DAPk-DD and the whole Ser-rich tail (residues 1293−1431) using the 5′-primer 5′-CCTGTATTT TCAGGGCGG GGAAGAAGA CCTG-3′ (forward) and 5′-ATCATCATC GAATTCTCA ACTCCTGTT CTGGAGG-3′ (reverse); second round PCR forward primer 5′-AATGATGAT GGATCCGAA AACCTGTAT TTTCAGGGC-3′. The final PCR product was ligated (BamHI and EcoRI) into pET-28GB1-1 (pET28a incorporating the GB1 coding sequence inserted between NdeI and BamHI sites). The resulting protein product is His6-(thrombin site)-GB1-(TEV site)-FADD-DD. The GB1-CD95-DD-His6 protein used in thermal denaturation experiments was kindly provided by Dr Diego Esposito .The template DNA used for the generation of the FADD-DD (residues 93−192) PCR insert was described previously The expression vector for full length human ERK2, encoded in the pGEX-6-P3 vector  as an N-terminal glutathione-S-transferase (GST)-tagged construct with a PreScission protease site, was a kind gift from Prof. Dario Alessi .E. coli BL21 codon plus cells (Stratagene). GB1-DAPk-DD proteins were expressed in the soluble phase and purified by Ni-IMAC and size-exclusion chromatography , followed by separation of the cleaved GB1 and TEV protease by passing the mixture through a Ni-NTA column.The expression and purification of the GB1-FADD-DD construct was performed as described above with minor modifications. FADD-DD was obtained by treating purified GB1-(TEV site)-FADD-DD with HisLarge scale production of recombinant ERK2 was carried out as previously described g for 10 min prior to injection to remove insoluble material. One-hundred microliter aliquots (0.1 to 20 mg ml−1) were sampled twice for each protein concentration. The flow rate was 1 ml.min−1 and experiments were run at room temperature. The column was calibrated using gel filtration molecular weight standards (Bio-Rad Laboratories). The protein standards were reconstituted in the same buffer used to equilibrate the column, but without dithiothreitol (DTT) to avoid disruption of disulfide bonds.Analytical size exclusion chromatography (SEC) experiments were performed using a pre-packed Superose 12 HR 10/30 column (∼24 ml bed volume) connected to an AKTA Purifier HPLC system  pre-equilibrated with the protein purification buffer. Elution profiles were recorded by monitoring the absorbance at 280 nm and elution volumes determined using the Unicorn software. Samples were centrifuged at 17,900 Circular dichroic (CD) spectra were acquired using a stopped-flow circular dichroism spectropolarimeter model 202SF (Aviv Instruments Inc.), with a CFT-23 recirculating chiller cooling unit (Neslab Instruments Inc.). Aviv CD v2.76 software was used to calculate the molar ellipticity. Spectra were recorded over the wavelength range of 190−260 nm. The path-length of the quartz cuvette was 0.1 mm. The bandwidth was 1 nm, settling time 0.3 sec, the averaging time 3 sec, and the wavelength step 0.5 nm. All samples were dialyzed overnight at 4°C. The protein sample concentration ranged from 10 to 50 µM and the CD spectra were normalized to yield mean residue ellipticity. All spectra were recorded at 25°C, unless otherwise stated. Samples were pre-equilibrated for 15 min prior to running the experiments. For each measurement three scans were averaged and subtracted from a ‘blank’ obtained for the buffer alone. The error bars plotted in the CD profiles represent an estimate of the uncertainty of the measurements derived from the three scans.−1 over the range 5–95°C. The reversibility of the thermal unfolding process was evaluated by recording the far UV spectrum upon cooling the protein to 5°C. The dynode voltage was monitored continuously to determine the useful isothermal wavelength range for spectral measurements and to evaluate the integrity of the protein samples during thermal denaturation. Where cooperative thermal unfolding was observed as a two-state process, the respective melting temperature (Tm) and free energy change (ΔGU→F) were derived by van’t Hoff analysis of the denaturation profiles.Thermal denaturation CD profiles were acquired by monitoring the ellipticity at 222 nm as a function of temperature employing a constant scan rate of 1 deg.min1H/15N/13C standard room temperature probe (600 MHz), or a HCN cold probe (800 MHz). Spectra were recorded at 25°C. Protein samples were prepared in 20 mM NaPi, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 7% D2O, pH 6.5. The protein concentration was 46 µM.NMR experiments were recorded on Varian Inova spectrometers (Agilent Technologies) operating at 600 and 800 MHz proton frequencies. The spectrometer was equipped with either a 5-mm z-gradient 1H−15N heteronuclear single quantum coherence (HSQC) spectra were recorded using a pulse-field gradient sensitivity-enhanced pulse scheme with the incorporation of water flip-back pulses. The data matrix consisted of 128*×384* data points (were n* refers to complex points) with acquisition times of 61 ms (tN) and 96 ms (tHN). A total of 32−128 scans per complex tN increment was collected. The recycle delay was 1.2 s. The total measuring time was between 1.1 and 4.5 h.Two-dimensional (2D) All raw NMR data were processed using nmrPipe Thermal denaturation experiments were recorded on an iQ5 Real-Time PCR Detection System controlled by the iQ5 optical system software version 1.0 (Bio-Rad Laboratories). Twenty-five microliters of a Sypro Orange 5×stock solution (Molecular Probes) were mixed with 25 µl of each protein sample to a final protein concentration of 50 µM. The fluorophore-mixed protein samples were loaded into an iQ5 96-well plate. The plate was heated from 25 to 85°C in increments of 0.5°C with a dwell time of 10 sec. Three samples for each different protein concentration were simultaneously assessed. The buffer conditions for GB1-DAPk-DD(L) were: 10 mM NaPi (pH 7.4), 150 mM NaCl, 0.5 mM DTT and 0.5 mM EDTA. The final buffer conditions for GB1-CD95-DD were: 5 mM Tris (pH 7.0), 25 mM NaCl, 0.5 mM EDTA and 1 mM β- mercaptoethanol.g for 6 min and equilibrated with 1 ml of the assay buffer. The assay buffer used was 50 mM HEPES (pH 7.5), 50 mM NaCl, 1% IGEPAL CA630, 20% glycerol, 10 mM EDTA and 1 mM DTT. Proteins used in the in vitro pull-down assays were derived from 5 ml LB cultures . Cell cultures were centrifuged and the cell pellets resuspended in 1 ml of the assay buffer. The cells were lysed by sonication and the lysates centrifuged at 17,900 g for 10 min. The cell supernatants were applied to the GST beads and incubated at 4°C for 2 h. Following centrifugation, cell lysates were decanted and the beads were washed seven times with 1 ml of the assay buffer. Fifty microliters of the elution buffer  were incubated with the beads for 10 min, centrifuged and the supernatant stored separately. The elution step was repeated twice and the three eluates were pooled.Fifty microliters (settled volume) of the GST beads (Novagen) were aliquoted into separate microcentrifuge tubes, centrifuged at 1,960 The His-tag monoclonal antibody (Novagen) and the accompanying colorimetric detection protocol were used for the Western blots. Incubation with the His-tag antibody was for 1 h and the blocking solution contained 5% milk. Seeblue2 protein markers (Invitrogen) were used for SDS-PAGE separation.n), enthalpy of interaction (ΔH), and the equilibrium dissociation constant (Kd). Data processing was performed with the Origin ITC analysis software package supplied by MicroCal.For isothermal titration calorimetry (ITC) measurements the protein samples were extensively dialyzed against 50 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM TCEP and 1 mM EDTA, and concentrated to the required level. Experiments were performed with a VP-ITC calorimeter : 19 aliquots of 15 µl of 159 µM GB1-DAPk-DD(L), or 250 µM of GB1-DAPk-DD(S), or 100 µM GB1 were injected into 1.45 ml of 8.85−18.6 µM ERK2 (different concentrations of ERK2 were employed for each titration) at 25°C. Experiments were repeated twice. Following subtraction of the corresponding heats of dilution, the binding isotherms were fitted using nonlinear least-squares optimization to obtain three parameters: binding stoichiometry , whereas SMART estimated the DAPk-DD to be longer at the amino terminus beginning at position 1300 and ending at position 1397 . In addition, a structure-based sequence alignment was performed and found to be in agreement with the sequence alignment. In this report, two DAPk-DD constructs are presented: DAPk-DD(S) (residues 1293−1399) and DAPk-DD(L) (1293−1408). Both constructs cover the putative DD boundaries defined by the bioinformatics analysis, therefore ensuring the inclusion of the complete core. Multiple strategies to express DAPk-DD as an isolated domain using overexpression in an E. coli system produced insoluble protein products. Attempts to refold the inclusion body material yielded negligible quantities of soluble product. To enhance the solubility we expressed DAPk-DD as a translational fusion protein at the C-terminus of the bacterial protein GB1, a well-characterized ‘solubility-enhancing tag’ that has been demonstrated to assist the recovery of soluble protein for a range of different proteins in E. coliA limited screening of the DD boundaries was performed. Boundaries tested were based on bioinformatics analysis of DD sequences and available structural data of DDs. The boundaries of the constructs prepared were derived based on Pfam, SMART and simple sequence alignments with other death domains. Pfam, in particular, predicted that the DAPk-DD to span residues 1312−1397  cis or in trans, and therefore must be a property of the DAPk-DD sequence itself. This finding is broadly consistent with the observation that DAPk-DD polypeptides, when expressed in isolation, spontaneously aggregate in inclusion bodies. Furthermore we can safely attribute the properties of the GB1-DAPk-DD fusion proteins in further biophysical analyses as arising from the sum of the (GB1 and DAPk-DD) parts.Since the protein samples were initially concentrated to 20 mg ml15N-1H HSQC NMR spectrum of isotope-labeled GB1-DAPk-DD(L) , whereas the corresponding value for DAPk-DD(L) is −10,250 deg.cm2.dmol−1. CD spectroscopy measurements have been previously reported for both FADD-DD and CD95-DD 2.dmol−1 respectively, in reasonable agreement to the values obtained here for FADD-DD. Similarly, the death domain of the Drosophila protein Pelle was reported to possess a CD spectrum showing double minima at 209 and 223 nm, with a MRE (223 nm) of ∼−18,000 deg cm2 dmol−1The CD spectrum of GB1-DAPk-DD(L) was recorded and corrected for the contribution from the GB1 domain . The ovePk-DD(L) . The spe–8.4 ppm . Analysi–8.4 ppm  and othe–8.4 ppm ; the speTm and ΔGU→F terms for the FADD-DD derived from a van’t Hoff analysis of the data are 335 K and −19.82 kJ mol−1respectively. The thermal denaturation profile of the GB1-corrected DAPk-DD profile features a very broad transition that is indicative of non-cooperative disruption of the α-helical structure and therefore not amenable to van’t Hoff analysis.To further assess the conformational status of the DAPk-DD we obtained the fluorimetric thermal denaturation profiles using the ‘Thermofluor’ method The conformational heterogeneity and apparent dynamic fluctuations of the DAPk-DD give rise to poor dispersion of the resonances in the NMR spectrum  that pre2.dmol−1) than in the absence of TFE, but lower when compared with the signal for the FADD-DD . The addition of 40% v/v TFE to the GB1-FADD-DD construct gave rise to a modest signal increase of 19% at 222 nm, suggesting that the helical content of this protein is closer to the maximum ‘capacity’ of the polypeptide sequence than for the DAPk-DD. The 2D 1H-15N HSQC spectrum of GB1-DAPk-DD(L) did not show any major change in signal dispersion in the presence of 40% v/v TFE  as a cosolvent has been shown to shift the conformational equilibrium of unfolded or molten globule-like proteins toward a stably-folded conformation suitable for NMR structural analysis  v/v TFE . There wRRDAADLLL) located within a region corresponding to H6 of the putative DAPk-DD fold in vitroIt has been reported that the putative DAPk-DD interacts with the extracellular signal-regulated kinases-1 and -2 (ERK 1/2) leading to modulation of the apoptotic activity of DAPk Unfortunately, the effect of the SR tail of DAPk towards ERK2 binding could not be fully investigated, because the constructs containing the SR tail  were expKd = 0.92 µM and 1.77 µM, respectively. In each case the observed stoichiometry (n) of the interaction is approximately 1∶1 (n = 1.03±0.01 (L) and 0.97±0.01 (S)). The corresponding ΔH, TΔS and ΔG values were −38.4 and −53.1 kJ mol−1, −4.0 and −20.3 kJ mol−1, and −34.4 and −32.8 kJ mol−1, for (L) and (S) constructs respectively. The isolated GB1 domain does not interact with ERK2 (data not shown). These results are consistent with the in vitro pull-down experiments showing that GB1-DAPk-DD(S) binds to ERK2 with an apparently lower affinity compared with the GB1-DAPk-DD(L) construct . The 2D 1H-15N HSQC spectrum shows very limited chemical shift dispersion for the great majority of resonances not attributable to the GB1 domain, suggesting an absence of globular structure for the DAPk-DD. Consistent with this observation we failed to detect a cooperative unfolding transition, similar to that observed for other GB1-proteins including the GB1-FADD-DD, during temperature-dependent thermal melting experiments monitored by fluorescence. Moreover the high level of Sypro Orange fluorescence observed in this experiment at the starting (low) temperature is consistent with the DAPk-DD protein component lacking ordered structure under ambient conditions. Therefore despite the essentially unambiguous bioinformatic assignment of a death domain fold for the DAPk-DD amino acid sequence, which appears both by visual inspection and quantitative measures of sequence similarity to sit well with other structurally-validated folded death domain proteins  towards ERK2-binding, suggesting an energy gain from the formation of intra- and/or intermolecular van der Waals contacts. This is accompanied by a significantly unfavorable entropic contribution (TΔS is negative) that, in combination with the relatively high exothermic ΔH values, is consistent with either the loss of a degree of flexibility or, more generally, a change in the conformation of one or both of the components in the DAPk-DD/ERK2 interaction.Despite the apparent lack of stable folding, we could demonstrate that the GB1-DAPk-DD(S) and (L) constructs are able to bind ERK2, consistent with reports that have also shown evidence for ERK2/DAPk-DD interactions both It is tempting to imagine that ERK2-binding might induce ordering of the DAPk-DD polypeptide, perhaps so far as to induce the formation of a canonical six-helix DD fold. We sought evidence for such an induced folding transition by recording CD spectra of the GST-ERK2/GB1-DAPk-DD(L) complex. However, to a good approximation the spectrum of the complex is essentially identical to the sum of the CD spectra of the individual components , suggest2-3-X2-6-ΦA-X-ΦB, where ΦA and ΦB are hydrophobic residues  A-X-ΦB portion of the ligand occupies a hydrophobic groove and the positively charged Arg/Lys residues interact with a negatively charged patch called the CD site A-X-ΦB ligands can bind without engagement of these charged residues The interaction of ERK1/2 with peptide ligands has been investigated in some detail 121-K-L-A-P-P-P-K-K129 which reads like a consensus D-motif peptide ligand except in reverse order. Indeed a variety of measurements suggests that this region of PEA-15 does bind to the ERK2 DRS ERK2 binding to the DED-containing protein PEA-15 has been investigated in some detail 1311-R-K-L-S-R-L-L-D1319 sequence near the N-terminus comes close to matching the D-motif consensus but lacks a hydrophobe at the ΦB residue (ΦB = Asp); the R1312-K-L-S-R-L-L-D-P1320 sequence is a close but non-exact match (ΦB = Pro) to the D-site consensus; separately, ΦA-X-ΦB D-‘subsite’ motifs occur at L1297-G-M1299, F1354-L-P1356, L1392-L-L1394 and I1402-N-L1404; and the sequence does not contain any amino acid stretch that is a close match to the F-site consensus. Thus the observed binding of DAPk-DD to ERK2 is likely attributable, at least in part, to an occupation of the DRS by one of the (non-exact) D-motifs that would be available within a disordered form of the DAPk-DD polypeptide. In fact, in the absence of an induced-fit mechanism, a globular DD-fold would, by homology, sequester the critical ΦA-X-ΦB hydrophobic residues in the core of the domain. However, neither the N- or C-terminal truncation mutants of DAPk-DD are competent to bind ERK2, therefore suggesting that binding must involve additional contacts outside of the putative DRS/D-motif interaction. This is reminiscent of the conclusions drawn for PEA-15 which is suggested to bind ERK2 in a ‘bidentate’ fashion How does this picture of ERK-ligand interactions apply to our observations with DAPk-DD? Our data clearly suggest that DAPk-DD does not function as a canonical death domain fold in order to bind to ERK2. This might suggest that the DAPk-DD could bind ERK2 via either the DRS or FRS, or both. Inspection of the amino acid sequence of the DAPk-DD constructs that we employed shows that: the Rholo-polypeptide. However, even in its partially helical, but mostly disordered, state the DAPk-DD is able to bind ERK2 with low micromolar affinity. Even though the binding does not involve induced folding of the DAPk-DD, it appears to require elements of the both the N- and C-terminal stretches of the protein. Collectively, these observations provide a platform for further investigation of the regulation of ERK1/2 and DAPk kinase activity in cell survival and death pathways at the molecular, cellular and biochemical level. Moreover emerging progress into the structural characterization of ERK2 by frontier methods in NMR spectroscopy in vitro or in vivo with other purported binding partners such as the netrin-1 receptor UNC5H2.In summary we have provided evidence that the death domain of DAPk is not stably folded in a classical, globular α-helical form, at least when expressed by itself out of the context of its ‘host’ Figure S1Protein purification by size-exclusion chromatography and analysis of the collected fractions by SDS-PAGE. (A) GB1-FADD-DD, GB1-DAPk-DD(S) and GB1-DAPk-DD(L) were purified by SEC on a Superdex 75 column  under similar conditions. The overlaid chromatograms clearly show a difference in profiles between GB1-FADD-DD and the two GB1-DAPk-DD constructs, as well as a difference in the elution volumes. The elution volume of the first peak is similar to the void volume and represents aggregated DAPk-DD species >70 kDa that do not dissociate under non-denaturing conditions. The second major peak represents oligomeric GB1-DAPk-DD species, which readily undergo reversible dissociation upon dilution to lower concentrations (see the Results section for further details and (C)). As a control, GB1-FADD-DD was purified using the same method and elutes at a later elution volume when compared with the elution volumes of the peaks arising from the GB1-DAPk-DD constructs. (B) SDS-PAGE analysis of GB1-DAPk-DD(S) fractions collected in (A). M: marker (MWs in kDa), A0: Ni/NTA eluate loaded onto the column, A6−A10: SEC fractions taken from (A). The gel bands were stained with Coomassie blue dye. (C) Affinity and SEC purified GB1-FADD-DD in 20 mM NaPi, 150 mM NaCl, 3 mM DTT, pH 6.2 and GB1-DAPk-DD(S) in 20 mM NaPi, 150 mM NaCl, 3 mM DTT, pH 7.4 were applied on a pre-packed Superose-12 analytical size-exclusion column (Amersham Biosciences) at the same concentration, i.e., 1 mg/ml. Coloured as in A. At a lower concentration, the purified GB1-DAPk-DD(S) protein elutes at an elution volume near identical to the GB1-FADD-DD protein, which does not form oligomers. This result shows that at the loading concentration of 1 mg/ml, the GB1-DAPk-DD(S) construct behaves as a monomer.(DOCX)Click here for additional data file.Figure S2Elution profiles of proteins studied by analytical size exclusion chromatography. Overlay of the different concentration runs on a Superose-12 column of ovalbumin (A), GB1-FADD-DD (C), GB1-DAPk-DD(S) (E) and GB1-DAPk-DD(L) (G) and zoomed in regions are presented in B, D, F and H, respectively. AU refers to the instrument absorbance units at 280 nm. Different concentrations in mg/ml from the highest to the lowest are as follow: blue (20), pink (10), red (5), cyan (1), purple (0.5), brown (0.25), green (0.1) and orange (0.05).(DOCX)Click here for additional data file.Figure S3Analytical gel filtration calibration curve. (A) Protein markers (Biorad) consisting of thyroglobulin (670 kDa), bovine gamma-globulin (158 kDa), chicken ovalbumin (44 kDa), equine myoglobin (17 kDa) and vitamin B12 (1.35 kDa) were run through the Superose-12 column. The order of the markers description follows the order of elution from left to right. The elution volumes of the protein markers, shown in ml next to the corresponding peaks, were calculated with the Unicorn 3.0 software package (Amersham Biosciences). Absorbance measured at 280 nm. Elution volumes related to the MWs  of the markers and the formula that correlates them are shown in (B). Diamond-shaped points in (B) represent the elution volumes derived in (A).(DOCX)Click here for additional data file.Figure S4CD spectra of DAPk-DD at 5, 25 and 95°C. CD spectra of GB1-corrected DAPk-DD(L) at 5°C (blue), 25°C (green) and 95°C (red). The CD spectra show no significant profile differences or signs of protein aggregation. A noticeable reduction in MRE values at the highest temperature is caused by the thermal denaturation of the secondary structure of the DAPk-DD. Error bars are estimates of uncertainties derived from the three scans recorded per sample.(DOCX)Click here for additional data file.Figure S5GB1-DAPk-DD shows little secondary structure change under different buffer conditions. Far-UV CD data of the GB1-subtracted native DAPk-DD(L) in different buffers. The effect of extreme pH  on the secondary structure of the DAPk-DD(L) is shown either in the presence of 150 mM NaCl (A) or in the absence of NaCl (B). Only at pH 2 in the absence of salt  did the DD show substantial changes in structure. (C) At pH 7.4, the influence of the detergent SDS was examined. Only minor differences were seen in the CD spectra recorded on GB1-DAPk-DD(L) samples measured in the absence of SDS (green), 2 mM  and 50 mM  SDS. The result indicates that SDS induced only slight secondary structure changes to the DD both below and above the CMC.(DOCX)Click here for additional data file.Figure S6The interaction of the DAPk-DD with ERK2 does not lead to any significant change in the secondary structure of the DAPk-DD or ERK2 proteins. CD analysis of the ERK-2-DAPk-DD complex. Far-UV CD spectra of ERK2 (blue circles) and GB1-corrected DAPk-DD(L) (green circles) are presented. The signal for the equimolar complex of GB1-DAPk-DD(L) and ERK2 was recorded (black circles), from which the ellipticity of GB1 was subtracted to give the native DAPk-DD-ERK2 CD signal. By combining the individual ERK2 and DAPK-DD CD data (red circles) a near identical CD profile was obtained to that observed for the complex, indicating negligible changes in secondary structure to either protein upon formation of a complex. The error bars are estimates of uncertainties derived from the three scans recorded per sample. The inset represents the data over the wavelength range of 200−230 nm.(DOCX)Click here for additional data file.


Escherichia coli, only a subset is associated with strains of extraintestinal pathogenic E. coli (ExPEC). These pathogens colonize diverse niches and are a major cause of sepsis, meningitis, and urinary tract infections. Using a murine infection model, we show that two TA systems (YefM-YoeB and YbaJ-Hha) independently promote colonization of the bladder by the reference uropathogenic ExPEC isolate CFT073, while a third TA system comprised of the toxin PasT and the antitoxin PasI is critical to ExPEC survival within the kidneys. The PasTI TA system also enhances ExPEC persister cell formation in the presence of antibiotics and markedly increases pathogen resistance to nutrient limitation as well as oxidative and nitrosative stresses. On its own, low-level expression of PasT protects ExPEC from these stresses, whereas overexpression of PasT is toxic and causes bacterial stasis. PasT-induced stasis can be rescued by overexpression of PasI, indicating that PasTI is a bona fide TA system. By mutagenesis, we find that the stress resistance and toxic effects of PasT can be uncoupled and mapped to distinct domains. Toxicity was specifically linked to sequences within the N-terminus of PasT, a region that also promotes the development of persister cells. These results indicate discrete, multipurpose functions for a TA-associated toxin and demonstrate that individual TA systems can provide bacteria with pronounced fitness advantages dependent on toxin expression levels and the specific environmental niche occupied.Toxin-antitoxin (TA) systems are prevalent in many bacterial genomes and have been implicated in biofilm and persister cell formation, but the contribution of individual chromosomally encoded TA systems during bacterial pathogenesis is not well understood. Of the known TA systems encoded by  E. coli (ExPEC). These bacteria are responsible for the majority of urinary tract infections worldwide, and are major causes of sepsis and meningitis. Using murine infection models with a reference uropathogenic ExPEC isolate, we found that three of the ExPEC-associated TA systems act independently to promote bacterial survival and persistence within the host urinary tract. Furthermore, we show that the toxin protein associated with one of these TA systems increases ExPEC stress resistance and persistence in the face of antibiotics. This work demonstrates the functional importance of specific TA systems to ExPEC pathogenesis, highlighting their potential as therapeutic targets.Toxin-antitoxin (TA) systems are widespread among prokaryotes, including many important human pathogens. It has long been hypothesized that TA systems contribute to bacterial pathogenesis, but clear-cut phenotypes associated with any individual TA system have not been described. Using bioinformatics, we demonstrate that distinct subsets of TA systems are linked with a major group of bacterial pathogens known as Extraintestinal Pathogenic  Toxin-antitoxin (TA) systems consist of stable toxic proteins that are held in check by co-expression of labile antitoxin molecules, the nature of which distinguishes three classes of TA systems Caulobacter crescentus genome are differentially expressed in response to heavy metal-, heat-, and nitric oxide-induced stresses Mycobacterium tuberculosis several TA systems are upregulated under hypoxic conditions or following bacterial entry into macrophages Environmental stresses have been shown to stimulate the transcription of multiple chromosomal TA loci, leading to the idea that these systems may enhance bacterial survival under adverse conditions E. coli at least 17 type II chromosomal TA systems have been identified in vitro to enhance survival of a K-12 E. coli strain in the presence of antibiotics by driving the formation of dormant, stress resistant cells known as persisters E. coli (ExPEC). These pathogens are able to colonize diverse niches within humans and other host animals and are a major cause of bacteremia, sepsis, peritonitis, neonatal meningitis, and urinary tract infections (UTIs) in vivo within the urinary tract. In addition, the toxin PasT encoded by one of these TA systems is shown to possess dual, concentration-dependent activities that either enhance bacterial stress resistance and growth or, alternatively, promote bacterial stasis and persister cell formation.Among strains of E. coli strains, the allelic profiles of 35 fully sequenced E. coli isolates were compared and organized based on similarities among 17 type II TA loci using the eBURST algorithm (Dataset S1) E. coli strains that differ in the number and composition of their type II TA systems (E. coli strains that includes most ExPEC isolates E. coli (EPEC) strain E2348/69 was also grouped with the ExPEC isolates. This is, however, in line with a previous report that classified this EPEC strain as a member of the B2 phylotype E. coli isolates can reflect their phylogenetic origin, suggesting that specific TA systems may serve an evolutionarily conserved purpose within this cohort.To ascertain potential patterns in the distribution and makeup of chromosomal type II TA systems among  systems . One of E. coli strains, we found that ExPEC generally have a reduced repertoire of intact type II TA loci, as exemplified by comparative analysis of the lab-adapted K-12 reference strain MG1655 and the ExPEC isolate CFT073 (E. coli (UPEC), the primary cause of UTIs worldwide yeeUV) are also present at sites around the CFT073 chromosome.Relative to K-12 e CFT073 . This pahigBA, ΔhipBA, and ΔsohA(prlF)-yhaV) tested showed no significant defects relative to the wild type strain (ybaJ-hha (tomB-hha) or the yefM-yoeB TA locus were clearly outcompeted by wild type CFT073 in the bladder, but not the kidneys (pasTI mutant (CFT073ΔpasTI) was outcompeted only in the kidneys . MG1655, which has a larger pool of TA systems, was much more adept at forming persisters than CFT073, and the deletion of pasTI did not affect the ability of MG1655 to form persisters in our assays , growth of the ΔpasTI mutant was significantly delayed  and acidified sodium nitrite (ASN), respectively  also resulted in small colony phenotypes as well as increased sensitivity to ASN  addition. Although no longer toxic, these PasT variants were still able to restore wild type growth of CFT073ΔpasTI on agar plates and in the presence of ASN . Furthermore, deletion of either yefM-yoeB or ybaJ-hha individually did not affect the development of persister cells in the face of ciprofloxacin or ampicillin  the ratA antitoxin encoded by Bacillus subtilisSalmonella species as well as many ExPEC isolates (e.g. NP_754911). Consequently, we refer to RatAB here as PasTI, for Persistence and stress-resistance Toxin and Immunity proteins.The PasT functioned like a toxin and inhibited bacterial growth in our assays only when expressed at high levels, similar to toxins within other TA systems. The toxic effects of PasT were completely reversible by induced expression of PasI, confirming that these two proteins can function as a TA pair . The toxE. coli isolates, ExPEC and other members of the B2 phylotype encode a condensed set of the known type II TA systems. E. coli strains are traditionally grouped within the B2 phylotype based in part on the presence of distinct virulence factors, such as fimbrial and toxin genes like papA, sfa/foc, hly, and cnf1E. coli isolates based solely on the makeup of their type II TA systems , following US federal guidelines indicated by the Office of Laboratory Animal Welfare (OLAW) and described in the Guide for the Care and Use of Laboratory Animals, 8th Edition.E. coli strains. These values were used to create an allelic profile for each fully sequenced E. coli isolate. The compiled allelic profiles were analyzed using the eBURST algorithm (http://eburst.mlst.net/) with 1,000 bootstrap iterations to organize strains into clusters within groups that share 17 out of 21 alleles Specific allelic values were assigned to indicate the presence, absence, or truncation of the known type II TA loci encoded by ATCTATTTATTTAGAGATCTGTTCTATTGTGATCTCTTATTAGGATCGCACTGCCCTGTGGATAACAAGGATCCGGCTTTTAAGATCAACAACCTGGAAAGGATCATTAACTGTGAATGATCGGTGATCCTGGACCGTATAAGCTGGGATCAGAATGAGGGGTTATACACAACTCAAAAACTGAACAACAGTTGTTCTTTGGATAACTACCGGTTGATCCAAGCTTCCTGA.Genomic locations of known type II TA systems encoded by MG1655 and CFT073 were obtained from NCBI and mapped onto circles that are proportional to the genome size of each strain. Origins of replication (OriC) were determined based on homology to “oriC” sequence: E. coli strains MG1655, CFT073, UTI89, F11, and S88 have been described previously tac promoter in the pRR48 backbone was induced by addition of 500 µM isopropyl-β-D-thiogalactopyranoside (IPTG), while gene expression from the pBAD33 promoter was induced using 0.2% L-arabinose. Primers and restriction sites used to construct all plasmids are indicated in All bacterial strains and plasmids used in this study are listed in higBA, hipBA, sohA-yhaV, ybaJ-hha, yefM-yoeB, or pasTI). PCR products were introduced via electroporation into the strains carrying pKM208, which encodes for an IPTG-inducible lambda red recombinase. Knockouts were confirmed by PCR using the primers listed in c3028 and c3029 to create CFT073-ClmR. This strain serves as a tagged wild type control in the gastrointestinal tract colonization assays.Targeted gene knockouts were generated in CFT073, MG1655, UTI89, F11, and S88 using the lambda Red-mediated linear transformation system 7 bacteria. CFT073 and isogenic knockout mutants were grown statically for 24 h in M9 medium, pelleted by spinning at 10,000 r.c.f. for 8 min, and resuspended in phosphate buffered saline (PBS) prior to inoculation. For competition assays, wild type and mutant strains were mixed 1∶1 prior to inoculation. The 1∶1 inoculation dosage was confirmed by plating serial dilutions on LB plates and LB plates containing 50 µg/mL kanamycin. Bladders and kidneys were recovered 1 or 3 d later, as indicated, and each was weighed and homogenized in 1 mL containing 0.025% Triton X-100. Homogenates were serially diluted and plated on LB agar plates to determine the number of bacteria per gram of tissue. For competition assays, plates containing kanamycin (50 µg/mL) were used to identify and enumerate the TA system knockout mutants, which carried KanR cassettes. Mouse experiments were repeated at least twice, and the total combined data from 10 or more animals is presented.Seven- to nine-week old female CBA/J mice (Jackson Labs), C3H/HeN mice (Harlan Laboratories), or C3H/HeJ mice (Jackson Labs) were used in accordance with IACUC-approved protocols as previously described R  and CFT073ΔpasTI (Rkan) grown 20 mL static in M9 broth were pelleted and resuspended in PBS. Adult female CBA/J mice were each gavaged with 50 µL of the bacterial suspension containing 1×109 CFU. Feces (∼100 mg) were collected at the indicated time points, weighed, and resuspended in 1 mL 0.7% NaCl. Serial dilutions were plated on LB agar containing either chloramphenicol (10 µg/mL) or kanamycin (50 µg/mL) in order to distinguish the ΔpasTI mutant and the ClmR-tagged strains. Mice receiving the CFT073 strains showed no overt signs of sickness. No chloramphenicol- or kanamycin-resistant bacteria were recovered in the feces of untreated mice.Cultures of CFT073-Clmin vivo competition assays were analyzed by Wilcoxon matched-pairs signed rank test. pasTI in the kidneys, were analyzed by Mann-Whitney two-tailed t tests. All statistical tests were performed using Prism 5.0c . P values less than 0.05 are considered significant.Persister assays were carried out with CFT073, MG1655, and their derivatives as previously described 2HPO4, 3 g/L KH2PO4, 1 g/L NH4Cl, 0.5 g/L NaCl, 1 mM MgSO4, 0.1 mM CaCl2, 0.1% glucose, 0.0025% nicotinic acid, 16.5 µg/mL thiamine, and 0.2% casein amino acids). Overnight cultures were diluted 1∶100 and growth of quadruplicate 200-µl samples in 100-well honeycomb plates was assessed at 37°C using a Bioscreen C instrument (Growth Curves USA). Bacteria assayed for growth in 1 mM acidified sodium nitrite (ASN) were first grown from frozen stocks overnight in MES-LB. Strains assayed for growth in more nutrient-limited, M9 medium supplemented with threonine  were initiated from cultures grown overnight in standard M9 medium. IPTG (500 µM) and 0.2% L-arabinose were added to cultures to induce high-level expression of PasT or PasI, as indicated. Methyl viologen  and ASN were prepared fresh prior to addition to LB or MES-LB broth cultures, respectively. All reagents were obtained from Sigma-Aldrich.Cultures of CFT073, MG1655 and their derivatives were grown shaking overnight at 37°C in 5 mL of LB broth, 100 mM morpholineethanesulfonic acid (MES)-buffered LB , or modified M9 minimal medium+casein amino acids  2; http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) The Cultures of each strain indicated were grown overnight shaking in LB broth plus 100 µg/mL ampicillin (added for plasmid retention). Cultures were diluted 1∶50 into fresh LB broth and grown shaking at 37°C for 5 h, at which point IPTG was added to each culture to a final concentration of 500 µM. After an additional 1 h incubation, 1 mL of each culture was pelleted and resuspended in 200 µL SDS-TE . Proteins were resolved by SDS-PAGE and transferred to Immobilon PVDF-FL membrane (Millipore). Western blots were probed using ANTI-FLAG M2 antibody (Sigma-Aldrich) and visualized by enhanced chemiluminescence as previously described Dataset S1Tables used to define the Type II TA system allelic profiles of E. coli by eBURST analysis. (Tab 1) Table showing TA system content among sequenced E. coli. (Tab 2) Table showing TA system content among phylotype B2 ExPEC strains. (Tab 3) Graph of the relative abundance of TA systems among all sequenced E. coli strains versus phylotype B2 isoaltes. (Tab 4) Table shows the individual allelic profiles of type II TA systems generated from Tab 1. (Tab 5) Table of allelic profiles that were used for input into the eBURST algorithm. (Tab 6) Separate tables with representative output from the eBURST algorithm, showing how the strains that are clustered together shift dependent upon based on the number of shared type II TA alleles considered.(XLS)Click here for additional data file.Figure S1The CFT073ΔyefM-yoeB and CFT073ΔybaJ-hha mutants colonize the murine urinary tract at similar levels to wild type CFT073 in noncompetitive assays. (A–D) Adult female C3H/HeN and C3H/HeJ mice were infected via catheterization with 107 CFU of wild type CFT073, CFT073ΔyefM-yoeB or CFT073ΔybaJ-hha. Graphs show bacterial titers present in the (A) bladders and (B) kidneys at 3 d post-inoculation. Bars indicate median values for each group; n≥10 mice. P values were determined using Mann-Whitney U tests.(TIF)Click here for additional data file.Figure S2CFT073 does not require pasTI for colonization of the murine gastrointestinal tract. Adult female CBA/J mice were each gavaged with 50 µL of a bacterial suspension containing 1×109 CFU CFT073-ClmR or CFT073ΔpasTI (Rkan). CFT073-ClmR served as the wild type control in these assays. Gastrointestinal tract colonization was assessed by enumerating total CFU of the mutant and wild type strains per gram of feces collected at the indicated time points. Data represent mean CFU/g feces ± SEM. n = 3 to 5 mice.(TIF)Click here for additional data file.Figure S3Low-level expression of PasT does not alter growth of the ΔpasTI mutant, while transcription of PasTI from its native promoter provides resistance to ASN. Curves show growth of CFT073 and its derivatives in (A) LB broth and (B) MES-LB broth+1 mM ASN. (A) Leaky expression of pasT from a Ptac promoter or expression of pasTI from its native promoter does not affect the growth of CFT073ΔpasTI in standard LB broth. In these assays, pRR48 served as an empty vector control. (B) Complementation of the ΔpasTI mutant growth defect in 1 mM ASN by the pasTI operon from its native promoter. Graphs are representative of at least two independent experiments performed in quadruplicate.(TIF)Click here for additional data file.Figure S4Persister cell formation by CFT073 requires the toxic domain of PasT. Graph shows numbers of viable bacteria (persisters) recovered 5 h after the addition of ciprofloxacin (10 µg/L) to broth cultures in exponential growth phase. The plasmid pRR48 serves as an empty vector control. Plasmid pPN007 encodes full-length PasT, pPN055 encodes the N-terminal 69 amino acids of PasT, and pPN068–069 encode non-toxic PasT variants lacking portions of the PasT N-terminus Click here for additional data file.Table S1Strains used in this study.(PDF)Click here for additional data file.Table S2Plasmids used in this study.(PDF)Click here for additional data file.Table S3Primers used in this study.(PDF)Click here for additional data file.


A highly divergent human papillomavirus (HPV) strain, HPV-L55, was identified in fecal samples from children hospitalized with diarrhea in China. The L1 gene of HPV-L55 shares <75% identity with previously reported HPVs, indicating that this virus represents a novel type of HPV. Phylogenetic analysis classified this virus as a member of the gammapapillomaviruses. Papillomaviridae  are a diverse class of small, circular, double-stranded DNA viruses in the family aviridae . Papilloomavirus . Recent omavirus , HPV-159omavirus , and HPVUsing the customized informatics pipeline VirusHunter , 7, we iThe full HPV-L55 genome was sequenced using the genome walking method . The primers were designed based on the original 454 pyrosequencing read, along with newly amplified HPV-L55 sequences; long fragments were further amplified using Ex Taq DNA polymerase  for final sequence confirmation. The sequences were then aligned and manually edited to produce the complete viral genome assembly. The complete circular genome of HPV-L55 is 7,277 bp in length with a G+C content of 37.47%.29CxxC separated by 36 amino acids in the E6 protein, and a modified zinc-binding domain CxC(x)29CxxC in the E7 protein  and a polyadenylation signal .The HPV-L55 genome encodes seven open reading frames (ORFs) , of which the putative E4 ORF contains a start codon and completely overlaps the E2 ORF. The genome contains a nucleotide-binding helicase domain (GPSNTGKS [G-X4-GKT/S]) in the E1 protein, two conserved zinc-binding domains of CxxC(x) protein , 9. In aGammapapillomavirus of Papillomaviridae, which currently includes >10 viral species known to infect humans . HPVs have traditionally been classified into different types based on the criterion that each type exhibits <90% similarity to the L1 nt sequence of other HPVs (Further studies will be necessary to determine the prevalence of this virus in the general population. Although a direct association between HPV-L55 and human disease has yet to be established, the full-genome sequence of HPV-L55 described here will be important for determining the full picture of genetic diversity in HPVs.KF482069.The full HPV-L55 genome sequence was deposited in GenBank under the accession no.


Brassica crops. Glucosinolates and their hydrolysis products are involved in plant–microbe, plant–insect, plant–animal, and plant–human interactions. It is, therefore, important to manipulate glucosinolate profiles and contents in Brassica species. In this study, aliphatic glucosinolates were genetically manipulated through homoeologous recombination in backcross lines followed by marker assisted selection in B. rapa. A resynthesized B. napus line, from a cross between B. rapa and B. oleracea, was backcrossed with Chinese cabbage doubled haploid line, RI16. Marker assisted selection for non-functional gene was performed in each backcross generations. Advanced backcross progenies (BC3F2) were developed to identify homoeologous gene replacement and/or introgression. Reduction in 5C aliphatic glucosinolates  was observed in BC3F2 progenies of the recurrent parent that carried the GSL-ELONG- gene. The GSL-ELONG- positive backcross progenies were also screened by the A-genome and BraGSL-ELONG gene specific marker, which linked with 5C aliphatic glucosinolates. The A-genome specific marker was absent in the plants of advanced backcross progenies which showed reduction in 5C aliphatic glucosinolates. The results suggest that the functional allele had been replaced by the non-functional GSL-ELONG- allele from B. oleracea. Some advanced backcross progenies (BC3F2) positive for the GSL-ELONG- allele and the A-genome specific SCAR marker BraMAM1-1 did not show reduction in 5C aliphatic glucosinolates, suggesting that GSL-ELONG- allele is recessive. Replacement of the functional locus in the A-genome by non-functional counterpart in the C-genome reduced the content of 5C aliphatic glucosinolates in B. rapa seeds with 20 μmol/g.Aliphatic glucosinolates are the predominant sulfur-rich plant secondary metabolites in economically important  Brassica, three diploid species, B. rapa L., B. oleracea L., and B. nigra L. Koch, are the evolutionary original genome donors of three amphidiploid species B. napus L., B. juncea L. Czern. & Coss., and B. carinata Br. These Brassica species are economically important crops and are cultivated for edible oil, industrial oil, and biodiesel. Since the introduction of Brassica species as crops in agricultural systems, many traits related to agronomy, morphology, physiology, yield, disease resistance, and end product quality have been improved. Most trait improvements have been achieved through conventional breeding approaches such as intraspecific and interspecific hybridization followed by selection. The development and analysis of alien chromosome addition lines were initiated in the 1990s for dissecting Brassica genomes for gene mapping and genetic analyses. Similarly, chromosome addition lines have also been deployed for the introgression of suitable traits from wild or weedy relatives to the existing cultivars. Alien chromosome addition monosomic and disomic lines have been successfully developed and proven to be useful resources for gene identification and chromosome homoeology studies between Brassica genomes of B. rapa × B. oleracea, B. napus × B. nigra, and B. rapa × B. oxyrrhina  gene located in the C-genome of B. napus, is reported to be involved in the regulation of non-homologous cross over during meiosis  are involved in side chain elongation, and a gene (BoGSL-ALK) is involved in side chain modification were identified and characterized in B. oleracea . Sequenced genome information has opened an avenue for comparative analysis of genome with Arabidopsis for glucosinolate biosynthesis genes. B. rapa, which revealed seven loci for BraGSL-ELONG genes for side chain elongation of aliphatic glucosinolates. Development and utilization of gene and/or loci specific molecular markers using sequenced genome would hasten marker assisted selection for improving quality traits like glucosinolates in Brassica vegetables and oilseeds.Glucosinolates, a class of nitrogen and sulfur containing plant secondary metabolites are found in thaliana . In B. roleracea . Li and inolates , 2003. MB. rapa through homoeologous gene replacement from B. oleracea (white cauliflower) using marker assisted selection. In this study, resynthesized B. napus lines were developed through interspecific hybridization between B. rapa and B. oleracea. Synthetic B. napus was backcrossed to B. rapa to develop B. rapa-B. oleracea chromosome addition lines harboring B. oleracea non-functional glucosinolate gene, GSL-ELONG- through marker assisted selection. The Brassica A-genome possesses at least four loci of the BraGSL-ELONG gene involved in 4C and 5C aliphatic glucosinolate biosynthesis  line, RI16 was crossed with a B. oleracea white cauliflower accession Snowball 76, and an embryo rescue technique was employed to produce resynthesized B. napus lines. The Chinese cabbage DH line, RI16 was derived from a hybrid cultivar, Summer Light 50 . One resynthesized B. napus line was backcrossed with B. rapa recurrent parent, Chinese cabbage (RI16) to produce backcross progenies. During recurrent backcrossing, marker assisted selection was performed in each generation with selection for genes of interest in the C-genome. In the first and second backcross cycle, recurrent parents were used as male parents. Additional reciprocal backcross was performed to enhance allosyndetical homoeologous recombination between the A-genome and alien chromosomes of B. oleracea. Advanced backcross progenies (BC3F1) were selfed to produce offspring homozygous for the replaced major glucosinolate gene GSL-ELONG- .A high glucosinolate content 2O.For screening and selection, DNA was extracted from leaf tissues of 2 week old seedlings using a modified cetyltrimethylammonium bromide (CTAB) method described by BoGSL-ELONG (GSL-ELONG-) gene in backcross progenies were performed using the C-genome specific SCAR markers. The polymerase chain reaction (PCR) was conducted using the primer pairs PM25 + PM13 and IPM8 + IPM7 for GSL-ELONG-(Table 1). Each PCR reaction of 10 μl consisted of 7.0 μl dH2O, 1.0 μl 10× PCR buffer, 0.3 μl 50 mM MgCl2, 0.15 μl 25 mM deoxyribonucleotide triphosphate (dNTP), 0.1 μl Taq polymerases, 0.15 μl 10 μM forward and reverse primers and 2.5 μl template DNA. The PCR program was 94°C for the initial 4 min to denature genomic DNA followed by 35 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 1.5 min. For all backcross generations, the GSL-ELONG- gene specific SCAR markers were scored on 1.2% agarose gels. One to seven plants from each family were selected using SCAR markers for the next backcross with the recurrent parents.Marker assisted selection of non-functional 1F1) and advanced (BC3F1) backcross progenies to determine glucosinolate profiles. Glucosinolate analysis was performed on BC3F2 seeds from the plants positive for the C-genome SCAR markers. Total glucosinolate content was extracted from 200 mg air dried seeds and 250 mg fresh young leaf tissues. Leaf and seed glucosinolate purification and overnight desulfation reactions were performed using sephadex  and purified sulfatase from Helix pomatia  as described by Leaf glucosinolate was extracted from early (BCTable 2).Leaf and seed desulfoglucosinolate separation and quantification was performed in a 5-μm column  coupled with the Alliance® reverse phase high-performance liquid chromatography  and photodiode array detector (Waters 996) system . Desulfoglucosinolates were separated using mobile phase HPLC grade methanol (A) and distilled water (B) at a flow rate of 1 ml/min. Both solvents were set up at gradients of an 8-min 7:93 A/B (v/v), a 4-min 15:85 A/B (v/v), an 18-min 55:45 A/B (v/v), a 5-min 92:8 A/B (v/v), a 5-min 92:8 A/B (v/v), a 5-min 1.5:98.5 A/B (v/v), a 3-min 1.5:98.5 A/B (v/v), and final 4-min 0:100 A/B (v/v) with total running time of 52 min. Individual glucosinolates were identified according to retention times and quantity was calculated in micromoles per gram and adjusted with relative response factors as described by BraGSL-ELONG gene specific SCAR markers (Table 1) for the glucosinolate side chain elongation were used to determine replacement of major effect locus/loci in B. rapa. Advanced backcross progenies were screened with the A-genome specific BraMAM1-1 and BraAE2 SCAR markers. These SCAR markers are dominant markers; therefore, absence of band was considered evidence of putative replacement/transposition of the native allele(s) with the corresponding allele from the C-genome.The A-genome and 3F3 plants from those lines which reduced 5C aliphatic glucosinolates. Flower buds were fixed for 24 h in propionic acid : absolute ethanol (1:3 v/v) and ferric chloride (~0.03%) was added as a mordant. Subsequently, buds were rinsed and stored in 70% ethanol. Flower buds were dissected and anthers were squashed in a drop of 1% acetocarmine on a glass slide. For chromosome counting, 10–20 pollen mother cells (PMCs) per flower bud, at least one bud per plant and two to four plants per lines were examined.Chromosomes were counts in BCB. napus line was developed by a cross between B. rapa genotype (high glucosinolate content) and B. oleracea white cauliflower genotype . The resynthesized B. napus line was backcrossed with B. rapa DH line RI16 of Chinese cabbage. The recurrent parent Chinese cabbage had distinct glucosinolate profile and content. The Chinese cabbage line produced gluconapin and trace amounts of progoitrin as 4C glucosinolates together with glucoalyssin and glucobrassicanapin as 5C aliphatic glucosinolates. Crossing of the resynthesized B. napus line with the B. rapa recurrent parent, RI16, on average, produced four seeds per silique (2–8), and resulted in digenomic triploid hybrid (AAC) plants. These AAC hybrid plants were subsequently backcrossed with the same B. rapa recurrent parent. In the case of the second backcross (BC2F1), on average, three seeds per silique (0–5) were recorded. Seed setting in BC2 hybrids were very low, about 40% silique did not produce seeds, apparently due to genomic irregularities in the plants. Positive plants (BC2F1) for the C-genome specific SCAR markers were selected and reciprocally backcrossed to the RI16 to produce BC3F1 seeds. In BC3F1 progenies, normal seed setting was observed, it could be due to reciprocal cross conducted that recovered recurrent genome and eliminated extra chromosomes from the genome. The C-genome specific SCAR marker positive plants (30%) were selfed to produce homozygous progenies (BC3F2). Seed setting was normal in all the homozygous progenies of BC3F1.A resynthesized GSL-ELONG- gene in marker assisted selection of backcross progenies . All backcross generations (BC1F1 to BC3F2) were screened with gene specific SCAR markers to obtain positive plants (Table 3). Marker positive plants in BC1F1 to BC3F1 could be due to the presence of the C-genome addition chromosome or due to the replacement or introgression of a C-genome fragment containing the GSL-ELONG- gene into the A-genome. In the backcross generations, loss of both the C-genome specific SCAR markers (PM25 + PM13 and IPM8 + IPM7) was observed in BC1 (six families), BC2 (four families), and BC3 (six families). The loss of these markers could be due to disappearance of additional chromosome without introgression and/or replacement events. On the other hand, four families in BC1, three families in BC2, and four families in BC3 were detected with the C-genome specific markers for GSL-ELONG- gene. This suggests that homoeologous recombination events might had occurred and resulted in replacement or introgression of the chromosomal fragments of the C-genome into the A-genome. There are three possibilities for the C-genome specific SCAR marker behavior, (i) replacement with the A-genome fragment, (ii) introgression of the C-genome fragment into the A-genome, and (iii) existence of additional chromosome in the backcross progenies (aneuploid). The A-genome specific SCAR markers for GSL-ELONG+ loci of chromosome A3 (BraMAM1-1) and chromosome A2 (BraAE2) were used to screen the BC3F2 population of the recurrent parent RI16 to investigate the occurrence of gene replacement or introgression. Fifteen BC3F2 plants of this population did not show the A-genome specific marker BraMAM1-1 for GSL-ELONG+ locus of chromosome A3, suggesting that homoeologous recombination and gene replacement had occurred. On the other hand, eight BC3F2 families displayed both the A- and C-genome specific markers, suggesting that introgression of GSL-ELONG- in the A-genome had occurred. However, no phenotypic changes in leaf and seed glucosinolates were observed in these families.The C-genome specific SCAR markers PM25 + PM13 and IPM8 + IPM7 were used to screen for B. rapa) containing 37 μmol/g seed 4C and 19 μmol/g seed 5C aliphatic glucosinolates and Snowball 76 (B. oleracea) containing 72 μmol/g seed 3C and trance amount of 4C and 5C aliphatic glucosinolates were used to develop resynthesized B. napus line containing 52 μmol/g seed 4C and 8 μmol/g seed 5C aliphatic glucosinolates. Total aliphatic glucosinolate contents in seeds of B. rapa (RI16), B. oleracea  and resynthesized B. napus line were 56, 74, and 60 μmol/g, respectively (Table 4).Two parental lines, RI16  were found to have reduced 5C aliphatic glucosinolates by five times to their recurrent parent RI16n and all these plants did not show the A-genome specific marker BraMAM1-1 corresponding to GSL-ELONG+ locus responsible for the biosynthesis of 5C aliphatic glucosinolates . On the other hand, 35 plants (70%) which were positive for the GSL-ELONG- marker did not show any changes in glucosinolate profiles due to the existence of corresponding functional alleles of the A-genome. The recurrent parent Chinese cabbage RI16 produced 20 μmole/g seed of 5C aliphatic glucosinolates, whereas some of the backcross progenies of RI16 produced trace amount (<5 μmol/g seed) of 5C aliphatic glucosinolates . The backcross progenies, which reduced 5C aliphatic glucosinolates were increased 4C aliphatic glucosinolates, resulting 4C glucosinolates represent total aliphatic glucosinolates in those progenies . Results of the A- and C-genome specific molecular markers and modification of glucosinolate profiles indicate possible functional gene replacement by non-functional alleles.Fifty BC3F3 plants which reduced 5C aliphatic glucosinolates. Two to four plants from each BC3F2 lines were analyzed for chromosome numbers. All PMCs showed normal cell divisions during meiosis and did not show any univalent or lagging chromosomes suggesting that all plants contained 20 chromosomes .Further to validate molecular marker results, chromosomes were counted in BCArabidopsis and Brassica species. B. rapa has a high level of genome duplication, which results in more loci with various functional properties (B. rapa have led to a very complex genetic basis for glucosinolate biosynthesis. B. rapa and B. oleracea diverged quite recently (approximately 7.9 MYA) from a common ancestor when compared to B. nigra (approximately between 16 and 18 MYA), therefore, relatively high homoeology exists between the A- and C-genomes  were observed in backcross progenies, this suggested variable homoeologous recombination events. This was in accordance with the previous studies reported in Brassica and other crop species. B. rapa × B. alboglabra. Raphanus sativus possessing monosomic additional chromosomes of B. rapa. Similarly, Allium monosomic addition lines. Chromosomal pairing and recombination between homoeologous chromosomes in digenomic triploid (AAC) lines of a occurs , 1987, ha occurs . Homoeola occurs . In thisB. napus, B. oleracea, or B. alboglabra with B. rapa  in B. rapa, respectively. In addition, comparative sequence analysis of Arabidopsis glucosinolate biosynthesis genes with the whole genome sequence dataset of B. rapa ssp. pekinensis cv. chiifu, B. rapa. Availability of genome and gene specific molecular markers with known gene function for glucosinolate biosynthesis pathway, breeding would be enhanced for specific glucosinolate profiles and contents in rapeseed and vegetable Brassica species. In the quantitative trait locus (QTL) mapping study for glucosinolates in B. rapa, Geng et al. (unpublished) identified a single major QTL for 5C aliphatic glucosinolates on linkage group A3 linked with the SCAR marker, BraMAM1-1. Our results are coherent with those findings and indicate that absence of BraMAM1-1 marker in the A-genome lack 5C aliphatic glucosinolates. This suggests that a major BraGSL-ELONG locus which elongates glucosinolate side chain for 5C aliphatic glucosinolate biosynthesis is located on chromosome A3 in the A-genome of B. rapa. On the other hand, no QTL detected on linkage group A9 suggested that BraGSL-ELONG locus on A9 may not play an important role in the biosynthesis of 5C aliphatic glucosinolates in the Chinese cabbage DH line, RI16 used in this study.Functional gene-based trait introgression has been reported in many studies of crosses between  B. rapa . HoweverFigure 5). Similar glucosinolate contents were reported by BnGSL-ELONG and Bn-GSL-ALK gene families in B. napus. These results suggested lack of functional genes at in some steps of chain reactions results in the accumulation of the precursors of glucosinolates in the biosynthesis pathway. For example, in side chain elongation, lack of functional BraGSL-ELONG locus/loci for 5C or 4C aliphatic glucosinolates would accumulate 4C or 3C aliphatic glucosinolates. In this study, other BraGSL-ELONG loci might have some roles in the biosynthesis of aliphatic glucosinolates including 5C aliphatic glucosinolates, which had not been replaced and resulted in variation of total aliphatic glucosinolate contents in different backcross families. The backcross progenies with random introgression of non-functional allele did not change aliphatic glucosinolate content.Glucosinolate profiles of 15 progenies which reduced 5C aliphatic glucosinolates also displayed increased in 4C aliphatic glucosinolates (GSL-ELONG- gene. This loss might be due to the random inclusion of the alien chromosome into the gametes during meiosis. Retention of the marker would then depend on the specific gametes involved during fertilization of backcrosses. Findings also reported loss of markers for QTL or genes in marker assisted backcross selection in crop species (GSL-ELONG- markers was observed. Similar studies reported for marker distortion to non-Mendelian segregation in Brassica and wheat (Loss of the markers for the alien chromosome or chromosomal fragment was observed in backcross progenies for  species . In advand wheat . This isGSL-ELONG- locus in these backcross progenies would be confirmed by additional backcrossing and sequencing the genomic regions of the replaced genes. Alternatively, resynthesized B. napus line would be developed through a cross between a B. rapa line with trace amount of 5C GSL and B. oleracea white cauliflower which lacks 4C and 5C aliphatic glucosinolates. The Chinese cabbage lines with reduced 5C aliphatic glucosinolates achieved in this study could be used for manipulation of glucosinolate profiles in B. napus, B. juncea, and other B. rapa accessions.Replacement or introgression of the 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.


A structure of a ribosome stalled during translation of the SecM peptide provides insight into the mechanism by which the large subunit active site is inactivated. Escherichia coli SecM (secretion monitor) gene product, which induces stalling to up-regulate translation initiation of the downstream secA gene, which is needed for protein export. Although many of the key components of SecM and the ribosomal tunnel have been identified, understanding of the mechanism by which the peptidyl transferase center of the ribosome is inactivated has been lacking. Here we present a cryo-electron microscopy reconstruction of a SecM-stalled ribosome nascent chain complex at 5.6 Å. While no cascade of rRNA conformational changes is evident, this structure reveals the direct interaction between critical residues of SecM and the ribosomal tunnel. Moreover, a shift in the position of the tRNA–nascent peptide linkage of the SecM-tRNA provides a rationale for peptidyl transferase center silencing, conditional on the simultaneous presence of a Pro-tRNAPro in the ribosomal A-site. These results suggest a distinct allosteric mechanism of regulating translational elongation by the SecM stalling peptide.As nascent polypeptide chains are synthesized, they pass through a tunnel in the large ribosomal subunit. Interaction between specific nascent chains and the ribosomal tunnel is used to induce translational stalling for the regulation of gene expression. One well-characterized example is the  Escherichia coli secretion monitor (SecM) polypeptide chain. The structure reveals the path of the SecM peptide through the tunnel as well as the sites of interaction with the tunnel components. Interestingly, the structure shows a shift in the position of the transfer RNA (tRNA) to which the growing SecM polypeptide chain is attached. Since peptide bond formation during protein synthesis requires precise placement of the substrates, namely, the peptidyl-tRNA and the incoming amino acyl-tRNA, it is proposed that this shift in the SecM-tRNA explains why peptide bond formation cannot occur and translation stalls.In all cells, ribosomes perform the job of making proteins. As the proteins are synthesized they pass through a tunnel in the ribosome, and some growing proteins interact with the tunnel, leading to stalling of protein synthesis. Here, we used cryo-electron microscopy to determine the structure of a ribosome stalled during the translation of the  The ribosome is a large macromolecular particle that synthesizes polypeptide chains from the substituent amino acid building blocks. The active site for peptide bond formation, the so-called peptidyl transferase center (PTC), is located in a cleft on the intersubunit side of the large ribosomal subunit  of the 170-aa SecM leader peptide is sufficient to induce translational arrest Gly) at the P-site and Pro-tRNAPro at the A-site of the ribosome Gly to the tRNAPro can still occur slowly In contrast to stalling by TnaC, translational stalling by SecM does not require an effector molecule Here we have determined a cryo-EM reconstruction of a SecM-stalled ribosome nascent chain complex (RNC) at 5.6 Å, enabling the direct interaction between critical residues of SecM and the ribosomal tunnel to be visualized. While we find no evidence for a cascade of rRNA conformational changes, we observe a shift in the position of the tRNA–nascent peptide linkage of the SecM-tRNA. This shift moves the carbonyl carbon of the SecM-tRNA away from the A-tRNA and, thus, is likely to contribute to the impaired activity of the PTC, explaining the SecM-mediated translational arrest.144–170) of SecM  wash protocol in our RNC preparation may provide an explanation for the low overall occupancy of A-site-bound Pro-tRNAPro (9%)  . DespitePro (9%) ; howeverPro (9%) . There istalling .Gly in the P-site to the A-site-bound Pro-tRNAPro  of ribosomes had undergone a ratchet-like subunit rearrangement of the small subunit relative to the large subunit . The rec-tRNAPro  and 3D. -tRNAPro . On this-Pro-RNC . The Sec-Pro-RNC .A molecular model for the SecM-stalled RNC was built by rigid-body docking of the ribosomal subunits from the model of the TnaC-stalled RNC The density characteristics indicate that the SecM nascent chain adopts a predominantly extended conformation, similar to that of TnaC 160QAQ158 area of SecM, are less likely to be important for SecM stalling  ;and reverse DP_SecM (5′-AATGGATTAGTGACAATAAAATTGAATTTACCCCACAAGCAAAATTCAGCACGCCCGTCTGGATAAGCCAGGCGCAAGGCATCCGTGCTGGCCCTCAACGCCTCACCTAATAA-3′) primers with DP120  construct as the template (E. coli in vitro translation system (Promega) programmed with SecM mRNA.The SecM construct was generated by PCR using forward T7_RBS_6xHis  and 0.1% pill/ml  at 70,000 g for 150 min in a TLA 120.2 rotor (Beckman Coulter) at 4°C. The supernatant  for 45 min at 4°C, transferred onto 500 µl of Talon Metal Affinity Resin (Clontech) pre-equilibrated with 250 buffer supplemented with 10 µg/ml of tRNAs and incubated for 5 min at room temperature. The resin was washed ten times with 1 ml of ice-cold 250 buffer. RNCs were eluted with 2.5 ml of 250 buffer supplemented with 100 mM imidazole (pH 7.0). The eluted RNCs were spun through 200 µl of a high salt sucrose cushion at 70,000 g for 150 min in a TLA 110 rotor at 4°C, and the resulting RNC pellet was resuspended in 1 ml of grid buffer  for 30 min at 4°C. The resulting SecM RNC  was further applied to 10 ml of sucrose on a 10%–40% gradient in 250 buffer in order to separate the monomeric SecM-stalled RNCs from the polysomes. Gradients were then centrifuged in a Beckman Coulter SW40-Ti rotor at 20,000 rpm for 4 h (4°C). In parallel, 1 ml of crude 70S ribosomes (2.5 OD260) prepared from the same extract used for translation was also applied on the sucrose gradient as a control  was applied to 2-nm carbon-coated holey grids. Micrographs were then recorded under low-dose conditions (25 electrons/Å2) with a magnification of 38,900 on a Tecnai F30 field emission gun electron microscope at 300 kV in a defocus range of 1.0–4.0 µm. Micrographs were scanned on a Heidelberg Primescan D8200 drum scanner, resulting in a pixel size of 1.24 Å on the object scale. The data were analyzed by determination of the contrast transfer function using CTFFIND software As described previously Haloarcula marismortui 50S subunit in complex with CCA-pcb http://www.pymol.org). All figures were generated using Chimera Densities for the 40S, 60S, and tRNAs were isolated using binary masks. Models were generated as described previously http://www.ebi.ac.uk/pdbe/emdb/) under accession numbers EMD-1829 and EMD-1830, respectively.The cryo-EM maps of the SecM-stalled RNC and SecM-Pro-RNC have been deposited in EMDataBank  SecM-stalled RNC, (B) SecM-stalled RNC with A-tRNA, and (C) SecM-Pro-RNC are 5.6 Å, 9.3 Å, and 6.0 Å, respectively, using the 0.5 FSC cutoff criterion.(0.15 MB TIF)Click here for additional data file.Figure S2Comparison of SecM-stalled RNCs. Reconstruction of the unsorted SecM-stalled RNC (gray) is compared (in boxed region) with reconstructions of (A) EMD-1143 (yellow) at approximately 15 Å (2.55 MB TIF)Click here for additional data file.Figure S3Conformational change in the SecM and SecM-Pro-RNCs. (A) Top (left) and side (right) views comparing SecM-Pro-RNC  with SecM-stalled RNC  aligned on the basis of the 50S subunit. Note the inward movement of the L1 stalk towards the P/E-tRNA (green) in the SecM-Pro-RNC as well as the ratcheting of the 30S subunit relative to the 50S. (B and C) Top (left) and side (right) views comparing (B) SecM-stalled RNC  or (C) SecM-Pro-RNC  with the hybrid state EMD-1541 (3.17 MB TIF)Click here for additional data file.Figure S4Visualization of the SecM nascent chain in the SecM-stalled RNC. (A–C) Transverse sections through (A) SecM-stalled RNC  at 5.6 Å, (B) EMD-1143 (yellow) at approximately 15 Å (1.31 MB TIF)Click here for additional data file.Figure S5Comparison of SecM and TnaC nascent chains within the tunnel. (A and B) Transverse sections through (A) SecM-stalled RNC  and (B) TnaC-stalled RNC (2.52 MB TIF)Click here for additional data file.Figure S6Comparison of SecM-stalled RNC filtered to different resolutions. Transverse sections through SecM-stalled RNC with SecM-tRNA in green and mRNA in red, filtered to (A) 6–7 Å, (B) 8–9 Å, and (C) 9–10 Å. Note the presence of small remaining density for the nascent chain in the upper tunnel but predominantly at the lower tunnel at 8–9 Å (B), indicating regions of compaction (1.19 MB TIF)Click here for additional data file.Figure S7Comparison of PTC of SecM-stalled and non-stalling peptide RNCs. Views of the PTC of the SecM-stalled-RNC alone (A and D) or compared with TnaC-stalled RNC (B and E) E. coli RNC with a non-stalling peptide at 7.1 Å (0.5 FSC) resolution (C and F) . Density for the SecM-stalled RNC is shown as gray surface in (A) and gray mesh in (D–F), with the model for the SecM-tRNA in green. Densities for the TnaC-stalled and non-stalling peptide RNCs are shown as yellow and blue surfaces in (B and E) and (C and F), respectively, with the molecular models for the peptidyl-tRNAs in gold and dark blue, respectively.(1.76 MB TIF)Click here for additional data file.Table S1Comparison of interactions of SecM and TnaC nascent chains with components of the ribosomal tunnel.(0.06 MB DOC)Click here for additional data file.