dataesr/scientific-paragraphs-categorization huggingface dataset:
pESBL also appears to encode factors that may promote its establishment in new hosts. For example, both R64 and pESBL encode ardA, which blocks the activity of the restriction endonuclease EcoK1  (22). Both plasmids also encode M. EcoGIX (referred to as YfbB in R64  (20) ), an unusual methyltransferase (MTase) that was recently shown to methylate only one of the double-strand DNAs, but whose biological significance remains unknown  (23). However, since DNA methylation is a common mechanism by which plasmids can escape digestion by host-encoded restriction endonucleases, the presence of these MTases may expand pESBL's host range. While there have been several studies of the transfer and replication machinery of R64 and ColIb-P9  (20, 24, 25) , knowledge of the regulatory processes that control the spread of this large group of plasmids remains incomplete and to date there have been no reports of the molecular factors that mediate and control spread of IncI1 plasmids that carry ESBLs, such as pESBL.
Most, but not all, of the genes that are required for pESBL transfer are important for R64 transfer as well. In addition to identifying pESBL loci that are required for its transfer, we found two very short regions in the plasmid that appear to regulate its transfer. Transposon-mediated disruption of these regions, whose precise roles remain to be elucidated, augments the levels of mRNA encoding pESBL transfer genes, suggesting that these loci encode a heretofore unrecognized regulatory system controlling spread of IncI1 plasmids. Lastly, we investigated the function of the pESBL-encoded M. EcoGIX MTase, which is also encoded by many IncI1 and IncF plasmids. This enzyme proved to protect pESBL from restriction in new hosts, suggesting it aids in the expansion of the host range of this plasmid.
0 platform for 65 cycles (Illumina). We prepared two independent libraries but the data analysis was done with the two data sets combined. Twenty four colonies from the output library were subjected to PCR with himmer3out and repZup primers to isolate mutants with Tn inserted in the Hft region. Two PCR-positive clones were chosen for characterization after the exact Tn insertion site was determined by sequencing. Oligonucleotides used for TnSeq are listed in Supplementary Table  S1.
The output of the program classified all loci within pESBL as either 'non-essential', 'essential' (i. e. genes essential for plasmid replication or maintenance), 'conditionally essentially' (i. e. genes required for transfer) or 'enriched' (genes that promote plasmid transfer). We utilized Con-ARTIST (37) to define genes that were conditionally essential for transfer. Briefly, the reads for each gene between the input and output libraries were compared using a Mann-Whitney U test. All insertions in genes that were significantly different (P-value <0. 002) were defined as conditionally essential and then fed into an HMM. The HMM refined the likelihood that each insertion was truly conditionally essential for transfer, essential for replication/maintenance or not required under either condition.
pESBL annotation Re-annotation of the pESBL genome was performed by a BLASTP search against the NCBI plasmid database (ftp://ftp. ncbi. nlm. nih. gov/genomes/Plasmids/) and KEGG database  (40). Coding DNA Sequences (CDSs) were annotated as a gene encodes a hypothetical protein when the CDSs had any of (i) E-value more than 1e-10, (ii) coverage of length less than 60% and (iii) sequence identity less than 40%, in the top hit of the BLASTP result. All the hypothetical genes were numbered for clarity. Furthermore, sogS, traK, pilK, repY and shufflon segments were added based on their identity to the R64 counterparts.
DNA sequences Accession numbers for the DNA sequences analyzed in this study are NC 018659. 1 (pESBL isolated from strain 2011C-3493) and AP005147. 1 (R64). We also referred to the pESBL sequence from E. coli O104:H4 strain TY-2482 found in the PATRIC database (http://patricbrc. org/portal/ portal/patric/Genome?cType=genome&cId=192592).
Additionally, the gene arrangement shown in this figure is a circular permutation of the arrangement listed in Table  2. Supplementary Figure  S1 ). Genes that are dispensable for transfer include bla-CTX-M-3 and bla-TEM, which encode the plasmid's extended spectrum beta lactamases, and most of pESBL's ORFs that lack significant homologs (annotated here as hypothetical proteins; hp) (Table  2 ). Most of these non-essential genes do not have counterparts in R64, suggesting that this region in R64-related plasmids may consist of variable 'cargo' genes that inserted into the core backbone of an ancestral plasmid during the evolution of this plasmid group. Notably, all 32 genes required for pESBL transfer correspond to parts of the four previously defined functional clusters within the R64 'transfer region'.
These clusters are (i) the nikAB and oriT region for conjugative DNA processing, (ii) the tra/trb gene cluster (spanning from traH -trbC) for general conjugation functions, (iii) the pil operon for type IV pilin biogenesis and (iv) the traABCD regulatory genes ((20) and Figure  2 ). With a few exceptions (outlined below), the genes we classified as essential for pESBL transfer (Table  2 ) were homologous to R64 genes found to mediate transfer in previous non-TnSeq-based analyses (24,44-48) (Figure  2 ). The pil genes, which encode the machinery for the biogenesis of a type IV pilus  (45) , appear to have somewhat distinct roles in R64 and pESBL. In R64, these genes are required for conjugative transfer in liquid media, but not on solid media  (24). However, in pESBL, five pil genes (pilRSTUV) were essential for pESBL transfer on the solid media we used for our screen.
Finally, we observed that none of the pESBL transcripts assayed were elevated for the Hft mutant, a difference in phenotype that provides further evidence that the transposon insertions do not simply result in loss of Hft function. Careful inspection of the distribution of insertions within the short (270 bp) Hft region revealed that the enriched reads in the output library were not equally distributed across this region. Instead, the enriched reads mapped to its ends, and bracket a middle zone with TA sites that were not enriched in the output library (Figure  3A ). This distribution provides additional support for the idea that the insertions may not all act in the same fashion and raises the possibility that the Hft region encodes more than a single activity, e. g. it mediates both activation and repression, and either activity can be modulated by transposon insertion.
The pESBL locus M. EcoGIX encodes an unusual single-strand DNA MTase activity with no conventional nucleotide recognition sequence  (23) , and homologs of this MTase can be found in many plasmids, e. g. YfbB of R64 (94% identity) and YfeA of F (91% identity) and in some chromosomes as well (Supplementary Table  S2 ). Our transposon insertion sequencing analyses revealed that M. EcoGIX is not re-quired for transmission of pESBL to E. coli CAG18439; however, CAG18439 does not encode a Type II restriction enzyme that targets sequences that might be methylated by M. EcoGIX. We hypothesized that introduction of DNA restriction endonucleases into recipient cells might reveal a role for M. EcoGIX during transmission, and to explore this possibility, we compared transmission of wild-type pESBL and pESBL lacking M.
Structure-solution problems Many data sets were collected from crystals of RpfC or the point mutations RpfC_E13A or RpfC_E13M with and without potential substrates and including selenomethionine-substituted RpfC_E13M at the ESRF, SLS, SOLEIL and Diamond synchrotrons. The automatic space-group assignment for all data sets gave the space group as C222 1 , with unit-cell parameters of around a = 66, b = 141, c = 90 A ˚, = = = 90. The resolutions of the data sets ranged from 3. 0 to 1. 9 A ˚. This would predict four copies of the RpfC chain in the asymmetric unit. We failed to obtain a molecular-replacement solution using our NMR structure (PDB entry 1xsf;  Cohen-Gonsaud et al. , 2005). Slightly better solutions were found using the crystal structure of the RpfB catalytic domain, with R and R free of around 0.
Structure analysis The asymmetric unit consists of eight copies of the RpfC chain. A set of four copies is generated by two twofold axes perpendicular to the crystallographic twofold; a single translation of (0. 554, 0. 0, 0. 109) then generates the second set of four copies (Fig. 1a ). Coupled with twinning the two folds give rise to the pseudo-C222 1 symmetry. Chains A, E and S have the most residues modelled into electron density (Gly1-Lys86) with an extra helix beyond the end of the conserved domain (Gly78). Chain B has the least modelled residues (Pro4-Gly78); the other chains are between these limits. We have modelled an ethylene glycol (the cryoprotectant) where a benzamidine molecule is present in the RpfB structures with PDB codes 4kpm  (Squeglia et al. , 2013)  and 4emn  (Ruggiero et al.
, 2013). As for the benzamidine in 4kpm, this is only seen in one of the similar interfaces. Benzamidine and ethylene glycol are not all that similar, but this observation indicates that this region in RPFs prefers binding small organic molecules to water. This region is part of the predicted binding site of a hexasaccharide based on superposition of the lysozyme-cleaved hexasaccharide complex with PDB code 1lzs  (Song et al. , 1994). The crystal packing of the two adjacent chains close to the benzamidine/ethylene glycol site is almost perfectly conserved in our RpfC structure and in the RpfB structures, despite there being no evidence of this contact being physiological. The two pairs of chain superimpose with an r. m. s. d. of 1. 1 A ˚over 149 residues using SSM  (Krissinel & Henrick, 2004) , which is not much larger than that for the single chains (see below).
The RPF domains are sufficiently close to clash with the superposed disaccharide in this region. The trisaccharide in 4kpm coincides with the other part of the cleaved saccharide in 1lzs (Fig. 1b ). As expected, the structural conservation between the new RpfC catalytic domain structure that we have determined in this study and the extensively studied RpfB domain is high. The calculated C r. m. s. d. between the two structures (our structure versus PDB entry 4kl7;  Squeglia et al. , 2013)  is only 0. 90 A ˚for 76 residues aligned by SSM with 52% sequence identity over the domain (Figs. 2a  and 2b ). Compared with the recent RpfE structure (PDB entry 4cge;  Mavrici et al. , 2014) , the calculated C r. m. s. d. is even lower at 0. 82 A ˚for 77 residues with 62% sequence identity (Figs. 1c  and 2a ).
indexing was carried out to set the angle to = 114 using the transformation matrix (100, 001, À110) with unit-cell parameters a = 65. 12, b = 88. 93, c = 157. 02 A ˚. Finally, the software REINDEX from CCP4 was used with settings h = h, k = k, l = l/2 to give the correct unit-cell lengths a = 65. 12, b = 88. 93, c = 78. 51 A ˚, = = 90, = 114. 50. The free set was then reduced to the P2 1 asymmetric unit and used as the source of free reflection flags for all other data sets. Initial phasing was carried out by MrBUMP (Keegan & Winn, 2008)  using the crystal structure of the catalytic domain of RpfB (PDB entry 3e05;.
This work was supported by grants from the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05-01 (MC-G), an X-TB Struct EU grant (MC-G and NHK), MRC grant G0401038 (GR, NHK, BH and JW), a Bloomsbury studentship to F-XC and a Commonwealth Studies Commission studentship MYCS-2011-252 to DHXQ.
So take a small circuit for the determinant (for instance the one given in  [20] ) and substitute its inputs by the entries of B. The result is a circuit C which computes a polynomial whose number of monomials is per(A). Observing that the determinant, and thus the polynomial computed by C, is multilinear completes the proof. for making this stay possible, thanks to funding from ANR ENUM (ANR-07-BLAN-0327). The third author would also like to thank his supervisor Peter Bürgisser for helpful advice. The type τ of a parse tree T for a circuit C is the function which associates to each edge e of C its multiplicity τ (e) in T. One could give an inductive characterization of parse tree types similar to the definition of parse trees above, but the following characterization is more useful. Add a unique artificial edge e out whose origin is the output gate of the circuit.
nepalensis (Pakistan), C. arborea (New Zealand), C. japonica (Japan), and C. microphylla (Mexico). [26]. Nodule lobes were crushed with sterile plastic mortars and pestles in 300 L of extraction buffer (100 mM Tris (pH 8), 20 mM EDTA, 1. 4 M NaCl, 2% (wt/vol) CTAB (cetyltrimethyl ammonium bromide), and 1% (wt/vol) PVPP (polyvinyl polypyrrolidone)). The homogenates were incubated at 65 ∘ C for 60min, extracted with chloroformisoamyl alcohol (24 : 1, vol/vol) and the resulting DNA was ethanol-precipitated and resolubilized. The extracted DNA was used for PCR amplification of both bacterial and plant DNA regions using the primers listed in Table  2. The amplicons were then cycle-sequenced in both directions using an ABI cycle sequencing kit (Applied Biosystem 3130). The nucleotide sequences obtained in this study were deposited in the NCBI nucleotide sequence database under the accession numbers given in Table  1.
Phylogenetic Analysis. Frankia strain CcI3 and Casuarina equisetifolia were used as outgroups in this study because they are physiologically distinct from the group studied yet phylogenetically close. The data sets were completed with homologous sequences present in the databases (Table  1 ). Alignments of Frankia glnA, dnaA, and IGS nif D-K and Coriaria matK and 18S rRNA-ITS1-5. 8S rRNA-ITS2-28S rRNA were generated with ClustalW  [27] , manually edited with MEGA 5. 0  [28]. Bacterial and plant sequences were separately concatenated and then used to examine maximum-likelihood cladogram evolutionary relationships of each symbiotic partner using 1000 bootstraps by following the GTR + G base substitution model. The distance between the sequences was calculated using Kimura's two-parameter model  [29].
Phylogenetic trees were constructed using the Neighbor-Joining method  [30]  with 1000 bootstraps  [31]  as implemented in MEGA 5. 0. In parallel, a Bayesian inference was realized with MrBayes  [32]  using the GTR + G model and 1,000,000 generations. A statistical test for the presence of congruence between Coriaria and Frankia phylogenies was evaluated through global distance-based fitting in ParaFit program  [33]  as implemented in CopyCat  [34]  and tests of random association were performed with 9999 permutations globally across both phylogenies for each association. An additional statistical test for correlation between geographical distances (obtained using http://www. daftlogic. com/projects-google-maps-distance-calculator. htm) and phylogenetic distances was made using Pearson's r correlation implemented in the R software  [35].
Acknowledgments This work is supported by  CMCU (Comité Mixte Tuniso-Francais pour la Coopération Inter-Universitaire  No. 10/G0903 ). The authors are grateful to  Dr. María Valdés  ( Escuela Nacional de Ciencias Biológicas, México, México ),  Dr. Sajjad Mirza  ( National Institute for Biotechnology Genetic Engineering, Faisalabad, Pakistan ),  Dr. Warwick Silvester  ( University of Waikato, Waikato, New Zealand ),  Dr. Kawther Benbrahim  and  Dr. A. Ennabili  ( University of Fes, Fes, Morocco ),  Mr. Spick  ( Montpellier Botanical garden, France ),  Dr. J. C. Cleyet-Marel  ( Montpellier INRA, France ),  Mr. D. Moukouanga  ( IRD Montpellier, France ), and  Dr. Takashi Yamanaka  ( Forest and Forestry Products Research Institute, Ibaraki, Japan ) for providing Coriaria nodules.
The data of temperature, wind and radiation were recorded by a data logger (CR3000, Campbell Scientific) every 10 s. A 3-D sonic anemometer (CSAT-3, Campbell Scientific) placed at z ref = 3. 0 m measured 3-D wind and temperature at 20 Hz and the data were recorded by a CR3000 data logger. The friction velocity (u * ) and stability functions (z/L) were computed using the TK3 software (see  Mauder and Foken, 2011). Eddy covariance fluxes of O 3 were simultaneously measured and are described in detail by  Plake et al. (2014).
Conclusions For the first time, we simultaneously measured transport times (aerodynamic resistances), vertical profiles of NO-NO 2 -O 3 mixing ratios and micrometeorological quantities within and above a natural grassland canopy. The obtained data were analysed to gain insights about the potential NO x canopy reduction in the grassland canopy, and to analyse the contribution of chemistry on fluxes of purely depositing compounds, such as O 3. We observed two extreme regimes: (a) high wind speed and low NO x mixing ratios (low NO x periods) and (b) low wind speed and high NO x mixing ratios (high NO x periods). Our study highlights that as a result of in-canopy convection, nighttime transport in the lowermost canopy layer is fastest, while conditions above the canopy are highly stable due to low wind speed during the high NO x periods.
Moreover, we determined a median net chemical O 3 production of 10 % during daytime within the air column between the EC flux measurement and the canopy, which was due to the absence of soil biogenic NO emission in our study. Hence, in contrast to previous studies our measured O 3 deposition flux by EC is slightly underestimated. The chemical flux divergence for O 3 was one order of magnitude larger during the high NO x than during the low NO x periods. Incanopy Damköhler numbers were shown to be relevant for NO 2 only under nighttime conditions, which was due to the minor role of NO 2 uptake by plants at this time. Damköhler numbers above the canopy indicated a potential flux divergence, but did not provide a hint for the observed chemical production of O 3. The only instance without indication of a chemical flux divergence within the entire data set was found during nighttime of the high NO x periods in the lowermost canopy layer.
Acknowledgements. This project was funded by the  Max Planck Society. We are grateful to  J. -C. Mayer  for the installation and the maintenance of meteorological sensors and for supporting the data evaluation. We are indebted to  A. Moravek  for his support with the eddy covariance measurements and the corresponding data evaluation and the fruitful discussions on many details of this study. The service charges for this open access publication have been covered by the  Max Planck Society. Edited by:  X. Wang.
54 to 4. 11 GHz indicated by the gray region in the figure. We can also distinguish partial gaps associated to each symmetry OO, EE, OE, or EO of the modes. The reason behind this symmetry distinction between the modes is important when searching for acoustic modes that can couple with the photonic modes OE studied here. To make this phoxonic structure more useful as an optomechanical tool, we have conducted a study over the design of a new phoxonic cavity capable of confining both electromagnetic and acoustic waves inside the gaps to enhance their interaction together with a high QF. Regarding the finite-element method, we used Comsol Multiphysics software to calculate the band structures and optical and acoustic wave fields of the trapped cavity modes. From these results, a script allows the calculation of coupling coefficients using the expressions reported by Chan et al.
Acknowledgements We express our gratitude to our colleagues in the CERN accelerator departments for the excellent performance of the LHC. We thank the technical and administrative staff at the LHCb institutes. We acknowledge support from  CERN  and from the national agencies:  CAPES ,  CNPq ,  FAPERJ  and  FINEP (Brazil) ;  MOST  and  NSFC (China) ;  CNRS/IN2P3.
035% UDM, further concentrated to 420 mg ml À 1 , and finally ultracentrifuged at 4360,000 g for 45 min. The protein was flash-frozen in liquid nitrogen and stored at À 80 °C. From a 10-l culture, highly purified MelB St at 30-40 mg was obtained routinely. Isothermal titration calorimetry. ITC measurements were performed in a nano isothermal titration calorimeter (TA Instruments). The purified MelB St in the dialysis buffer was injected into the ITC sample cell and 6 mM melibiose dissolved in the same dialysis buffer was titrated incrementally into the protein sample, and the heat rate was recorded at 25 °C. Melibiose binding was also measured in the presence of 100 mM LiCl. The cumulative heat change (DQ) was plotted against the molar ratio of melibiose to MelB St and fitted with the one-site independent binding model (NanoAnalyse software).
5 ) for D 2 G FRET. Applying the same experimental setup, NaCl was consecutively added to the sample containing the RSO vesicles and D 2 G (10 mM)  9. An identical volume of water was used for the control. Fluorescent increase (DI, the difference before (I 0 ) and after the addition of NaCl) was expressed as the percentage of the I 0 , corrected by a dilution effect, and then plotted as a function of Na þ concentration. The K 0. 5 for Na þ simulation was determined by fitting a hyperbolic function to the data (OriginPro). Mutagenesis. All MelB St mutants were constructed with the QuickChange Site-Directed Mutagenesis Kits (Stratagene) using the plasmid pK95 DAH/MelB St / CHis 10 as the template  4. Gel filtration chromatography. Gel filtration was performed in a Superdex 200 10/300 GL column on a Fast Protein Liquid Chromatography instrument.
5), 100 mM NaCl, 50 mM CaCl 2 , 35-37% PEG 400 and 0. 08% octyl-b-D-galactopyranoside. Crystals were frozen with liquid nitrogen and tested for X-ray diffraction at the Lawrence Berkeley National Laboratory, ALS BL 8. 2. 1, 8. 2. 2, 5. 0. 1, 5. 0. 2 or at the Stanford Synchrotron Radiation Light Source BL 7-1 via remote data collection. The complete diffraction data set was collected at 100 K from a single cryocooled crystal at wavelength of 1. 004 Å with an ADSC QUANTUM 315 detector at ALS BL 5. 0. 2; image data were processed with HKL 2000 (ref. 61) to a resolution of 3. 35 Å with 98% completeness (Table  1 ). Structure solution and refinement. Data were processed in space groups P3 2 21, P3 1 21, P3 2 and P3 1. The data set was analysed by Structure Factor Check on CCP4 (ref. 62) suite and Phenix Xtriage  63, 64  , which indicated a strong off-origin peak at 0, 0, 0.
Final refinement was carried out using the Tangent Least-Square Jelly-Body-Restrained twin refinement, where the R/R free values dropped to 0. 30/0. 35 (Table  1 ). As pseudosymmetries are present, higher apparent R/R free values are expected  67. Out of 476 residues, 1-432 in Mol-A, regions 1-210 and 235-450 in Mol-B, 1-431 in Mol-C, as well as 1-100, 109-210, 235-398 and 408-431 in Mol-D, were well resolved. Residues (82. 2%) are in most favoured, 17. 4% in generously allowed and 0. 4% in disallowed regions. Visualization of omit maps and manual model building were performed using Coot 0. 7 (ref. 68). Surface electropotential maps were calculated using APBS software  69. All crystallographic figures were generated with Pymol 1. 5 (ref. 70).
Acknowledgements We thank  Luis Reuss  and  R. Bryan Sutton  for critical reading of the manuscript, and  Shailika Nurva ,  Hariharan Parameswaran ,  Elena Tikhonova ,  Gill Verner  and  Shushi Nagamori  for assistance. We thank  Kelsey Markham  for constructing most of the mutants. We also thank the staffs at the  Advanced Light Source, Lawrence Berkeley National Laboratory  and the  Stanford Synchrotron Radiation Light Source  for assistance with X-ray data collection. The diffraction data set was collected at ALS BL 5. 0. 2. This work was supported by the  National Science Foundation  (grant  MCB-1158085  to L. G. and grant  MCB-0450970  to H. R. K. ) and the  Norman Hackerman Advanced Research Program  (grant  010674-0034-2009  to L. G. ), as well as by the  National Institutes of Health  (grants  R21HL087895  and  R01GM095538  to L.