Jorgelina Morán-Barrio

María-Natalia Lisa

Nicole Larrieux

Salvador I Drusin

Alejandro M Viale

Diego M Moreno

Alejandro Buschiazzo

Alejandro J Vila
email: vila@ibr-conicet.gov.ar

CRYSTAL STRUCTURE OF THE METALLO-BETA-LACTAMASE GOB IN THE PERIPLASMIC DI-ZINC FORM REVEALS AN UNUSUAL METAL SITE

Metallo-beta-lactamases (MBLs) are broad spectrum, Zn(II) dependent lactamases able to confer resistance to virtually every β-lactam antibiotic currently available. The large diversity of active site structures and metal content among MBLs from different sources has limited the design of a pan-MBL inhibitor. GOB-18 is a divergent MBL from subclass B3, expressed by the opportunistic Gram-negative pathogen Elizabethkingia meningoseptica. This MBL is atypical since several residues conserved in B3 enzymes (such as a metal ligand His) are substituted in GOB enzymes. Here we report the crystal structure of the periplasmic di-Zn(II) form of GOB-18.

This enzyme displays a unique active site structure, with residue Gln116 coordinating the Zn1 ion through its terminal amide moiety, replacing a ubiquitous His residue. This situation contrasts with that of B2 MBLs, where an equivalent His116Asn substitution leads to a di-Zn(II) inactive species. Instead, both the mono-and di-Zn(II) forms of GOB-18 are active against penicillins, cephalosporins and carbapenems. In silico docking and molecular dynamics simulations indicate that residue Met221 is not involved in substrate binding, in contrast with Ser221, otherwise conserved in most B3 enzymes. These distinctive features are conserved in recently reported GOB orthologues in environmental bacteria. These findings provide valuable information for inhibitor design, and also posit that GOB enzymes might have alternative functions.

INTRODUCTION

The expression of β-lactamases is the main mechanism of bacterial resistance against βlactam antibiotics. These enzymes catalyze the hydrolysis of the amide bond in the β-lactam ring characteristic of this family of drugs [START_REF] Meini | Overcoming differences: The catalytic mechanism of metallo-beta-lactamases[END_REF][START_REF] Meini | Evolution of Metallo-beta-lactamases: Trends Revealed by Natural Diversity and in vitro Evolution[END_REF][START_REF] Palzkill | Metallo-beta-lactamase structure and function[END_REF][START_REF] Llarrull | The future of the betalactams[END_REF][START_REF] Fisher | Bacterial resistance to beta-lactam antibiotics: compelling opportunism, compelling opportunity[END_REF]. MBLs are metal-dependent hydrolases which generally use Zn(II) as a Lewis acid to activate a water molecule for the nucleophilic attack.

These enzymes are refractive to clinically employed lactamase inhibitors [START_REF] Meini | Overcoming differences: The catalytic mechanism of metallo-beta-lactamases[END_REF] and have a particular relevance in the clinical setting as they can hydrolyze a broad spectrum of β-lactam substrates, being able to inactivate carbapenems, the "last resort" antibiotics in antibacterial therapy [START_REF] Crowder | Metallo-beta-lactamases: Novel Weaponry for Antibiotic Resistance in Bacteria[END_REF].

MBLs have been classified into subclasses B1, B2 and B3, based on sequence identity [START_REF] Galleni | Standard numbering scheme for class B beta-lactamases[END_REF].

Crystal structures of MBLs from the three subclasses have revealed that these enzymes present a common αβ/βα sandwich fold, with the active site located within a groove at the interface between these two halves [START_REF] Meini | Overcoming differences: The catalytic mechanism of metallo-beta-lactamases[END_REF][START_REF] Meini | Evolution of Metallo-beta-lactamases: Trends Revealed by Natural Diversity and in vitro Evolution[END_REF][START_REF] Palzkill | Metallo-beta-lactamase structure and function[END_REF][START_REF] Llarrull | The future of the betalactams[END_REF][START_REF] Fisher | Bacterial resistance to beta-lactam antibiotics: compelling opportunism, compelling opportunity[END_REF][START_REF] Crowder | Metallo-beta-lactamases: Novel Weaponry for Antibiotic Resistance in Bacteria[END_REF]. The Zn(II)-binding residues vary among different subclasses, giving rise to diverse metal site architectures and metal contents required for activity [START_REF] Meini | Overcoming differences: The catalytic mechanism of metallo-beta-lactamases[END_REF][START_REF] Meini | Evolution of Metallo-beta-lactamases: Trends Revealed by Natural Diversity and in vitro Evolution[END_REF][START_REF] Palzkill | Metallo-beta-lactamase structure and function[END_REF][START_REF] Llarrull | The future of the betalactams[END_REF][START_REF] Fisher | Bacterial resistance to beta-lactam antibiotics: compelling opportunism, compelling opportunity[END_REF][START_REF] Crowder | Metallo-beta-lactamases: Novel Weaponry for Antibiotic Resistance in Bacteria[END_REF]. B1 and B3 MBLs are broad-spectrum enzymes that hydrolyze penicillins, cephalosporins, and carbapenems with a wide variety of in vitro catalytic efficiencies, displaying a broad range of resistance profiles in vivo [START_REF] Meini | Overcoming differences: The catalytic mechanism of metallo-beta-lactamases[END_REF][START_REF] Meini | Evolution of Metallo-beta-lactamases: Trends Revealed by Natural Diversity and in vitro Evolution[END_REF][START_REF] Palzkill | Metallo-beta-lactamase structure and function[END_REF][START_REF] Llarrull | The future of the betalactams[END_REF][START_REF] Fisher | Bacterial resistance to beta-lactam antibiotics: compelling opportunism, compelling opportunity[END_REF][START_REF] Gonzalez | Metallobeta-lactamases withstand low Zn(II) conditions by tuning metal-ligand interactions[END_REF]. The di-Zn(II) form of B1 MBLs has been shown to be the active form in the bacterial periplasm, despite contradictory data obtained from in vitro studies [START_REF] Gonzalez | Metallobeta-lactamases withstand low Zn(II) conditions by tuning metal-ligand interactions[END_REF][START_REF] Llarrull | Metal content and localization during turnover in B. cereus metallo-beta-lactamase[END_REF][START_REF] Hawk | Differential binding of Co(II) and Zn(II) to metallo-betalactamase Bla2 from Bacillus anthracis[END_REF].

These enzymes display a conserved metal binding motif, where the coordination sphere of the metal ion in the Zn1 site involves three His residues (3-H site), His116, His118 and His196, and a water/hydroxide molecule (the active nucleophile) in a tetrahedral arrangement, while the on September 21, 2018 by guest http://aac.asm.org/ Downloaded from metal ion in the Zn2 site adopts a trigonal bipyramidal coordination sphere, with Asp120, Cys221, His263 (DCH site), a water molecule, and the formerly mentioned water/hydroxide molecule as ligands [START_REF] Fabiane | Crystal structure of the zinc-dependent beta lactamase from Bacillus cereus at 1.9 A resolution: binuclear active site with features of a mononuclear enzime[END_REF][START_REF] Garcia-Saez | The three-dimensional structure of VIM-2, a Zn-beta-lactamase from Pseudomonas aeruginosa in its reduced and oxidised form[END_REF][START_REF] Concha | Crystal structure of the widespectrum binuclear zinc beta-lactamase from Bacteroides fragilis[END_REF][START_REF] King | New Delhi metallo-betalactamase: structural insights into beta-lactam recognition and inhibition[END_REF][START_REF] Gonzalez | Host-specific enzymesubstrate interactions in SPM-1 metallo-beta-lactamase are modulated by second sphere residues[END_REF] (Figure 1). Instead, B2 MBLs are essentially exclusive carbapenemases and are active with one Zn(II) ion bound to the Zn2 site (superimposable to the B1 DCH site), while binding of a second metal equivalent to the Zn1 site results in enzyme inhibition [START_REF] Bebrone | The structure of the dizinc subclass B2 metallobeta-lactamase CphA reveals that the second inhibitory zinc ion binds in the histidine site[END_REF][START_REF] Hernandez | Zn(II) dependence of the Aeromonas hydrophila AE036 metallo-beta-lactamase activity and stability[END_REF]. Substitution His116Asn (present in all B2 enzymes) impairs metal binding to the Zn1 site by removing a metal ligand. Consequently, nucleophile activation does not involve a metal ion [START_REF] Bebrone | The structure of the dizinc subclass B2 metallobeta-lactamase CphA reveals that the second inhibitory zinc ion binds in the histidine site[END_REF][START_REF] Fonseca | Crystal structure of Serratia fonticola Sfh-I: activation of the nucleophile in mono-zinc metallo-betalactamases[END_REF][START_REF] Garau | A metallobeta-lactamase enzyme in action: crystal structures of the monozinc carbapenemase CphA and its complex with biapenem[END_REF]. B3 lactamases, distantly related to the B1 and B2 subclasses, are mostly di-Zn(II) enzymes [START_REF] Ullah | The crystal strucuture of the L1 metallo-beta-lactamase from Stenotrophomonas maltophilia at 1.7 A resolution[END_REF][START_REF] Garcia-Saez | Three-dimensional structure of FEZ-1, a monomeric subclass B3 metallo-beta-lactamase from Fluoribacter gormanii, in native form and in complex with D-captopril[END_REF][START_REF] Docquier | High-resolution crystal structure of the subclass B3 metallo-betalactamase BJP-1: rational basis for substrate specificity and interaction with sulfonamides[END_REF][START_REF] Wachino | Structural insights into the subclass B3 metallo-beta-lactamase SMB-1 and the mode of on September 21, 2018 by guest http[END_REF][START_REF] Leiros | Crystal structure of the mobile metallo-beta-lactamase AIM-1 from Pseudomonas aeruginosa: insights into antibiotic binding and the role of Gln157[END_REF]. The Zn1 site of B3 enzymes is a 3-H site similar to that of B1 enzymes, whereas two mutations (Cys221Ser and Arg121His) affect the Zn2 coordination geometry [START_REF] Ullah | The crystal strucuture of the L1 metallo-beta-lactamase from Stenotrophomonas maltophilia at 1.7 A resolution[END_REF][START_REF] Garcia-Saez | Three-dimensional structure of FEZ-1, a monomeric subclass B3 metallo-beta-lactamase from Fluoribacter gormanii, in native form and in complex with D-captopril[END_REF][START_REF] Docquier | High-resolution crystal structure of the subclass B3 metallo-betalactamase BJP-1: rational basis for substrate specificity and interaction with sulfonamides[END_REF][START_REF] Wachino | Structural insights into the subclass B3 metallo-beta-lactamase SMB-1 and the mode of on September 21, 2018 by guest http[END_REF][START_REF] Leiros | Crystal structure of the mobile metallo-beta-lactamase AIM-1 from Pseudomonas aeruginosa: insights into antibiotic binding and the role of Gln157[END_REF], and the metal ion is bonded to Asp120, His121 and His263. Ultimately, Ser221 (equivalent to Cys221 in the DCH site) is no longer a metal ligand, so that the Zn2 site is a DHH site in B3 MBLs. Besides, a third water molecule participates as a fifth ligand, giving rise to a square pyramidal coordination sphere of Zn2 [START_REF] Ullah | The crystal strucuture of the L1 metallo-beta-lactamase from Stenotrophomonas maltophilia at 1.7 A resolution[END_REF][START_REF] Garcia-Saez | Three-dimensional structure of FEZ-1, a monomeric subclass B3 metallo-beta-lactamase from Fluoribacter gormanii, in native form and in complex with D-captopril[END_REF][START_REF] Docquier | High-resolution crystal structure of the subclass B3 metallo-betalactamase BJP-1: rational basis for substrate specificity and interaction with sulfonamides[END_REF][START_REF] Wachino | Structural insights into the subclass B3 metallo-beta-lactamase SMB-1 and the mode of on September 21, 2018 by guest http[END_REF][START_REF] Leiros | Crystal structure of the mobile metallo-beta-lactamase AIM-1 from Pseudomonas aeruginosa: insights into antibiotic binding and the role of Gln157[END_REF]. Similarly to B1 enzymes, the Zn1 ion in B3 lactamases is regarded as responsible of nucleophile activation [START_REF] Ullah | The crystal strucuture of the L1 metallo-beta-lactamase from Stenotrophomonas maltophilia at 1.7 A resolution[END_REF][START_REF] Garcia-Saez | Three-dimensional structure of FEZ-1, a monomeric subclass B3 metallo-beta-lactamase from Fluoribacter gormanii, in native form and in complex with D-captopril[END_REF][START_REF] Docquier | High-resolution crystal structure of the subclass B3 metallo-betalactamase BJP-1: rational basis for substrate specificity and interaction with sulfonamides[END_REF][START_REF] Wachino | Structural insights into the subclass B3 metallo-beta-lactamase SMB-1 and the mode of on September 21, 2018 by guest http[END_REF][START_REF] Leiros | Crystal structure of the mobile metallo-beta-lactamase AIM-1 from Pseudomonas aeruginosa: insights into antibiotic binding and the role of Gln157[END_REF].

The deepest branching member of the MBL B3 subclass, GOB from Elizabethkingia meningoseptica (formerly Chryseobacterium meningosepticum) presents several unusual features. Despite being related to B3 lactamases based on sequence homology, GOB enzymes present a His116Gln substitution that resembles the active site mutation found in B2 enzymes [START_REF] Bellais | Molecular and biochemical heterogeneity of class B carbapenem-hydrolyzing beta-lactamases in Chryseobacterium meningosepticum[END_REF]. In addition, Ser221 is replaced by a Met residue, suggesting a more divergent metal binding site, or a B2-B3 hybrid enzyme. GOB-type enzymes include 18 allelic variants, all of them expressed by E. meningoseptica, a pathogen responsible for neonatal meningitis and on September 21, 2018 by guest http://aac.asm.org/ Downloaded from opportunistic infections in immunocompromised patients [START_REF] Bloch | Chryseobacterium meningosepticum: an emerging pathogen among immunocompromised adults. Report of 6 cases and literature review[END_REF][START_REF] Lee | Chryseobacterium meningosepticum sepsis complicated with retroperitoneal hematoma and pleural effusion in a diabetic patient[END_REF][START_REF] Shinha | Bacteremia due to Elizabethkingia meningoseptica[END_REF]. So far, two GOB variants have been characterized biochemically: GOB-1 and GOB-18 [START_REF] Moran-Barrio | The metallo-beta-lactamase GOB Is a mono-Zn(II) enzyme with a novel active site[END_REF][START_REF] Horsfall | Broad antibiotic resistance profile of the subclass B3 metallo-beta-lactamase GOB-1, a di-zinc enzyme[END_REF][START_REF] Moran-Barrio | In vivo impact of Met221 substitution in GOB metallo-beta-lactamase[END_REF][START_REF] Lisa | Probing the Role of Met221 in the Unusual Metallo-beta-lactamase GOB-18[END_REF], which differ by three residues located far from the active site [START_REF] Moran-Barrio | The metallo-beta-lactamase GOB Is a mono-Zn(II) enzyme with a novel active site[END_REF]. Expression of GOB-1 in the periplasm of E. coli led to a di-Zn(II) enzyme showing a broad substrate profile, similar to most B3 MBLs [START_REF] Horsfall | Broad antibiotic resistance profile of the subclass B3 metallo-beta-lactamase GOB-1, a di-zinc enzyme[END_REF]. Instead, cytoplasmic expression of GOB-18 resulted in accumulation of a non-active Fe(III)-substituted form. This variant, when metal depleted and reconstituted with Zn(II), was able to bind only one metal equivalent in the putative DHH site, as confirmed by mutagenesis and spectroscopic studies [START_REF] Moran-Barrio | The metallo-beta-lactamase GOB Is a mono-Zn(II) enzyme with a novel active site[END_REF]. Besides, mono-Zn(II)-GOB-18 behaved as a fully active, broad spectrum B3 enzyme. These data, together with the unusual residues present in positions 116 and 221, suggested that GOB enzymes might have novel structural and functional features.

Here we report the crystal structure of the periplasmic form of GOB-18, which shows that GOB enzymes are unique among MBLs. GOB-18 contains a dinuclear Zn(II) site, revealing that replacement of His116 does not preclude these lactamases to bind two metal ions. Indeed, Gln116 is a metal-ligand in the Zn1 site, resulting in a novel HQH site instead of the canonical 3-H site. Met221 (yet another singularity of GOB enzymes) is not a metal ligand and is not involved in substrate binding, further confirmed by docking and molecular dynamics simulations. Thus, GOB enzymes present a divergent metal binding site with novel substrate binding features, which make them unique among B3 MBLs. This observation, together with the finding that the resistance profile of E. meningoseptica is elicited mostly by the B1 enzyme BlaB [START_REF] Gonzalez | Carbapenem resistance in Elizabethkingia meningoseptica is mediated by metallo-beta-lactamase BlaB[END_REF], discloses a functional redundancy, and suggests that GOB enzymes may play alternative roles in this microorganism yet to be disclosed.

MATERIALS AND METHODS

Chemicals.

Biochemical reagents were purchased from Sigma-Aldrich except when specified.

Molecular biology reagents were purchased from Promega, Invitrogen or New England Biolabs.

Metal-free buffers were prepared adding Chelex 100 to normal buffers and stirring for 0.5 h.

Production of recombinant GOB-18 in the periplasmic space of E. coli. GOB-18 was produced in

the periplasmic space of E. coli C41 (DE3) cells harboring the plasmid pKP-GOB-18 [START_REF] Moran-Barrio | The metallo-beta-lactamase GOB Is a mono-Zn(II) enzyme with a novel active site[END_REF][START_REF] Moran-Barrio | In vivo impact of Met221 substitution in GOB metallo-beta-lactamase[END_REF][START_REF] Lisa | Probing the Role of Met221 in the Unusual Metallo-beta-lactamase GOB-18[END_REF]. This vector allows the production of recombinant GOB-18 as a C-terminal fusion to the leader peptide pelB [START_REF] Moran-Barrio | The metallo-beta-lactamase GOB Is a mono-Zn(II) enzyme with a novel active site[END_REF][START_REF] Moran-Barrio | In vivo impact of Met221 substitution in GOB metallo-beta-lactamase[END_REF]. Secretion via the Sec pathway allows processing of the precursor and targeting the mature protein to the periplasmic space of the bacterial host. E. coli C41 (DE3) cells harboring plasmid pKP-GOB-18 were grown aerobically at 30ºC in LB broth supplemented with kanamycin (50 µg/ml) until reaching 0.6 units of absorbance at 600 nm. Protein production was induced adding isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM, the incubation was continued for 16 h at the same temperature. Mature GOB-18 was extracted from the cellular periplasm by a two-step osmotic shock procedure without lysozyme nor EDTA. Briefly, cells were harvested and resuspended with 1 ml/g (wet weight) of 20 mM Tris-HCl pH 8, 50% w/v sucrose, 0.1 mM phenylmethylsulfonyl fluoride, and incubated for 2 h at 4ºC under orbital shaking. 10 ml of ice-cold water were added per ml of suspension, and further incubated for 2 h in the same conditions. After centrifugation at 15,000 g for 8 min at 4ºC, the supernatant (first extract), was collected and stored at 4ºC. The pellet was further resuspended in ice-cold water in half the volume used for the first extract, and incubated in the same on September 21, 2018 by guest http://aac.asm.org/ Downloaded from conditions for 1.5 h at 4ºC. The supernatant (second extract) was collected and pooled with the first one rendering the periplasmic fraction.

Purification of GOB-18 from the periplasmic fraction of E. coli. Mature GOB-18 was purified to homogeneity with two steps of ionic exchange chromatography (Figure 2). The periplasmic extract was adjusted to pH 7 and loaded onto a Sephadex CM-50 column (Sigma) preequilibrated with buffer E (20 mM Tris-HCl, pH 7). The resin was washed with 5 column volumes of buffer E, and eluted with buffer E supplemented with 400 mM NaCl. The elution peak was concentrated, dialyzed against buffer E, and loaded onto a Mono S column (GE Healthcare) preequilibrated with buffer E. Elution was achieved with buffer E with added NaCl (gradient 0 to 1 M). Samples containing GOB-18 were pooled and concentrated to 20 mg/ml, frozen in liquid nitrogen and stored at -70ºC until crystallogenesis assays. An aliquot of the enzyme was stored at 4ºC for kinetic analyses. The average purification yields obtained were ca. 1 mg of GOB-18 per liter of E. coli culture, resulting in the expected 31 kDa polypeptide, as estimated by SDS-PAGE.

Metal content determination.

The metal content of purified GOB-18 was measured by atomic absorption spectroscopy in a Metrolab 250 instrument operating in the flame mode.

Circular dichroism spectroscopy.

The circular dichroism spectrum of periplasmic GOB-18 was obtained using a JASCO J-810 spectropolarimeter at 25ºC. on September 21, 2018 by guest http://aac.asm.org/ Downloaded from Determination of the kinetic parameters. Antibiotic hydrolysis was monitored by absorbance variation resulting from the scission of the β-lactam ring. Reactions were performed in 15 mM Hepes pH 7.5, 200 mM NaCl at 30ºC. The kinetic parameters K M and k cat were derived from initial rate measurements, as recorded with a Jasco V-550 spectrophotometer, and were estimated by nonlinear data fitting to the integrated form of the Michaelis-Menten equation.

Crystallization and data collection. Crystals grew from a 9.8 mg/ml solution of GOB-18, by adding 0.5 µl of protein to 1.5 µl mother liquor (100 mM Tris-HCl pH 8.5, 1.7 M NaCl, 1.7 M (NH 4 ) 2 SO 4 ) in a hanging-drop setup with 1 ml mother liquor in the reservoir. These were not single crystals, which were eventually obtained by microseeding fresh drops with identical conditions except for using a slightly modified mother liquor (2 M NaCl/ (NH 4 ) 2 SO 4 and avoiding Tris-HCl). Single crystals reached a size of ca. 150 µm, and were cryoprotected in mother liquor containing 25% glycerol and flash frozen in liquid nitrogen. X-ray diffraction data were collected in house (Unit of Protein Crystallography, Institut Pasteur de Montevideo, Montevideo, Uruguay), from a single crystal, with a MicroMax-007 HF X-ray source (Rigaku) and a MAR345 image plate detector (Mar Research), employing radiation of 1.5418 Å. The diffraction data were processed using XDS [START_REF] Kabsch | XDS[END_REF] and scaled with Aimless (35) from the CCP4 program suite.

Structure determination and refinement. The crystal structure of GOB-18 was solved by molecular replacement using the program Phaser [START_REF] Mccoy | Phaser crystallographic software[END_REF] imipenem was built in silico by making a structural alignment with the structure of L1-imipenem using the Multiseq plug-in of VMD [START_REF] Humphrey | VMD: visual molecular dynamics[END_REF]. Molecular dynamics simulations were performed starting from complexes GOB-18-imipenem and L1-imipenem. Each complex was immersed in a truncated octahedral periodic box with a minimum solute-wall distance of 8 Å, filled with explicit TIP3P water molecules [START_REF] Jorgensen | Comparison of simple potential functions for simulationg liquid water[END_REF]. Molecular dynamic simulations were performed with the AMBER14 package [START_REF] Pearlman | AMBER, a package of computer programs for applying molecular mechanics, normal mode analysis, molecular dynamics and free energy calculations to simulate the structural and energetic properties of molecules[END_REF][START_REF] Case | AMBER 14[END_REF], using ff14SB [START_REF] Maier | ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB[END_REF]. Particle-mesh Ewald (PME) was implemented for long range interactions with a cutoff distance of 12 Å (48). Temperature and pressure were regulated with the Berendsen thermostat and barostat [START_REF] Berendsen | Molecular dynamics with coupling to an external bath[END_REF]. All bonds involving hydrogen were on September 21, 2018 by guest http://aac.asm.org/ Downloaded from fixed using the SHAKE algorithm (50). Each initial system was equilibrated at 300K using a conventional protocol, and then subjected to 50 ns of simulation in the NVT ensemble. To maintain the coordination environment around zinc atoms we used harmonic bonds between the metal centers and the residues of the coordination sphere. We applied a restraint to keep the imipenem bound to the active site. Root mean squared deviation (rmsd) and root mean square fluctuations (rmsf) calculations of the molecular dynamics simulations were performed using the cpptraj module of AMBER [START_REF] Roe | PTRAJ and CPPTRAJ: software for processing and analysis of molecular synamics trajectory data[END_REF].

on September 21, 2018 by guest http://aac.asm.org/ Downloaded from

RESULTS

Expression and purification of GOB-18 for crystallogenesis

Production of GOB-18 in E. coli gives rise to different metallated species depending on the cellular compartment where the protein accumulates. Overexpression of GOB-18 in the cytoplasm results in an inactive Fe(III)-bound form, from which a mononuclear fully-active Zn(II)-variant can be prepared by metal chelation and remetallation in vitro [START_REF] Moran-Barrio | The metallo-beta-lactamase GOB Is a mono-Zn(II) enzyme with a novel active site[END_REF]. Instead, GOB binds exclusively Zn(II) when it is secreted into the bacterial periplasm [START_REF] Moran-Barrio | The metallo-beta-lactamase GOB Is a mono-Zn(II) enzyme with a novel active site[END_REF][START_REF] Moran-Barrio | In vivo impact of Met221 substitution in GOB metallo-beta-lactamase[END_REF]. GOB-18 produced in the cytoplasm of E. coli, both the Fe(III)-containing variant or the reconstituted mono-Zn(II) form, proved recalcitrant after extensive crystallization trials. An alternative protocol was thus optimized allowing to produce GOB-18 in the bacterial periplasm, which is the physiological cellular compartment for MBLs' expression in Gram-negative species (Figure 2A). This strategy yielded GOB-18 containing exclusively Zn(II), with a maximum metal content of (1.4± 0.1) Zn(II) equivalents/ GOB-18 molecule as determined by atomic absorption spectroscopy. This metal-content figure indicates the presence of a dinuclear GOB-18 species, which is consistent with previous reports for GOB-1 [START_REF] Horsfall | Broad antibiotic resistance profile of the subclass B3 metallo-beta-lactamase GOB-1, a di-zinc enzyme[END_REF]. Circular dichroism of periplasmic GOB-18 indicates it has similar tertiary structure as the in vitro reconstituted Zn(II) form (29) (Figure 2B). Periplasmic GOB-18 was able to catalyze the hydrolysis of a broad spectrum of β-lactam substrates with k cat and K M values similar to those formerly reported for mono-Zn(II) and di-Zn(II)-GOB-1, with no changes in the catalytic efficiencies upon addition of 20 μM Zn(II) to the reaction medium (Table 1). Nevertheless, in contrast to reconstituted Zn(II)-GOB-18, on September 21, 2018 by guest http://aac.asm.org/ Downloaded from periplasmic GOB-18 crystallized in different conditions containing high concentrations of NaCl and (NH 4 ) 2 SO 4 as precipitants.

Crystal structure of periplasmic GOB-18

Periplasmic GOB-18 crystallized in space group P2 1 , with crystals diffracting X rays to 2.6 Å resolution (Table 2). The final refined atomic model contains two protein molecules per asymmetric unit, 4 zinc atoms, 8 chlorides, 77 waters and one glycerol molecule. The two protein chains are very similar, with 0.26 Å root mean squared deviation (rmsd) among 266 aligned α-carbons. Chain A includes residues Ser17 to Asp286 (the side chains of residues Asn26 and Asp286 were not included in the model due to weak electron density), while monomer B spans residues Val20 to Lys290 (similarly, Val20 side chain was not modeled), with a continuous main chain trace throughout. GOB-18 displays the expected αβ/βα sandwich fold, with two core β-sheets composed of 7 (β1-β7) and 5 (β8-β12) β-strands, respectively, and 6 α-helices (Figure 3A). Helices α1-α4 cover the 7-stranded β-sheet, with helix α4 closing one side of the metal-binding groove. On the other hand, helices α5 and α6 wrap around the 5-stranded βsheet, with the intervening region β12-α6, including three short helical elements, closing up the other side of the catalytic site. As already described for the MBL fold (1-6), the whole domain of GOB-18 can be depicted as a duplication of two structurally similar α/β hemidomains. GOB-18 is very similar to other B3 lactamases [START_REF] Ullah | The crystal strucuture of the L1 metallo-beta-lactamase from Stenotrophomonas maltophilia at 1.7 A resolution[END_REF][START_REF] Garcia-Saez | Three-dimensional structure of FEZ-1, a monomeric subclass B3 metallo-beta-lactamase from Fluoribacter gormanii, in native form and in complex with D-captopril[END_REF][START_REF] Docquier | High-resolution crystal structure of the subclass B3 metallo-betalactamase BJP-1: rational basis for substrate specificity and interaction with sulfonamides[END_REF][START_REF] Wachino | Structural insights into the subclass B3 metallo-beta-lactamase SMB-1 and the mode of on September 21, 2018 by guest http[END_REF][START_REF] Leiros | Crystal structure of the mobile metallo-beta-lactamase AIM-1 from Pseudomonas aeruginosa: insights into antibiotic binding and the role of Gln157[END_REF], despite low sequence identity.

Structural alignment of GOB-18 (chain A) with available B3 lactamase models allows for similarity quantification (Figure 3B): FEZ-1 (PDB 1K07, 1.27 Å rmsd for 243 aligned residues); on September 21, 2018 by guest http://aac.asm.org/ Downloaded from BJP-1 (PDB 3LVZ, 1.50 Å rmsd for 239 residues); and, L1 (PDB 1SML, 1.55 Å rmsd for 236 residues). When calculated per residue, the largest local rmsd values are observed for two loops flanking the active site, which are expected to be involved in substrate specificity: loop 1 (residues 130-152 in GOB-18, equivalent to 148-172 according to BBL numbering; to facilitate comparative analyses, residue numbering will hereafter be stated for GOB-18 followed by BBL numbering in parenthesis) and loop 2 (198-218(219-239)). Structural differences are also noticeable in the β12-α6 loop (residues 239-265(261-287)) as well as in the N-terminus of the protein.

All B3 lactamases crystallized so far are characterized by at least one disulfide bridge, in most cases between Cys residues located in the C-terminal helix and the loop linking elements α5 and β12 [START_REF] Ullah | The crystal strucuture of the L1 metallo-beta-lactamase from Stenotrophomonas maltophilia at 1.7 A resolution[END_REF][START_REF] Garcia-Saez | Three-dimensional structure of FEZ-1, a monomeric subclass B3 metallo-beta-lactamase from Fluoribacter gormanii, in native form and in complex with D-captopril[END_REF][START_REF] Docquier | High-resolution crystal structure of the subclass B3 metallo-betalactamase BJP-1: rational basis for substrate specificity and interaction with sulfonamides[END_REF][START_REF] Wachino | Structural insights into the subclass B3 metallo-beta-lactamase SMB-1 and the mode of on September 21, 2018 by guest http[END_REF][START_REF] Leiros | Crystal structure of the mobile metallo-beta-lactamase AIM-1 from Pseudomonas aeruginosa: insights into antibiotic binding and the role of Gln157[END_REF]. GOB-18 is an exception in this regard within B3 enzymes, with only one Cys residue in its primary structure, i.e. Cys180(201) (in strand β10), which is buried within the protein core (Figure 3A) forming hydrogen bonds in the base of the metal binding site (see below).

The active site of di-Zn(II)-GOB-18

The electron density maps revealed the presence of two heavy atoms in the active sites of each GOB-18 monomer in the asymmetric unit (Figure 4A). They occupy the metal binding sites found in other dinuclear MBLs, and can be recognized as the Zn1 and Zn2 ions. The Zn-Zn distance (3.5 and 3.8 Å in chains A and B, respectively) is similar to that found in other dinuclear B3 lactamases [START_REF] Ullah | The crystal strucuture of the L1 metallo-beta-lactamase from Stenotrophomonas maltophilia at 1.7 A resolution[END_REF][START_REF] Garcia-Saez | Three-dimensional structure of FEZ-1, a monomeric subclass B3 metallo-beta-lactamase from Fluoribacter gormanii, in native form and in complex with D-captopril[END_REF][START_REF] Docquier | High-resolution crystal structure of the subclass B3 metallo-betalactamase BJP-1: rational basis for substrate specificity and interaction with sulfonamides[END_REF][START_REF] Wachino | Structural insights into the subclass B3 metallo-beta-lactamase SMB-1 and the mode of on September 21, 2018 by guest http[END_REF][START_REF] Leiros | Crystal structure of the mobile metallo-beta-lactamase AIM-1 from Pseudomonas aeruginosa: insights into antibiotic binding and the role of Gln157[END_REF]. Zn1 is coordinated by the Nδ1 atom of residue His100(118), His175(196) Nε2, Gln98(116) Oε1 and a Zn-Zn bridging water molecule. The Zn1-Oε1(Gln) distance is 2.0 Å, on September 21, 2018 by guest http://aac.asm.org/ Downloaded from pinpointing Gln98(116) as a novel metal-binding residue in MBLs. On the other hand, Zn2 is coordinated to His103(121) and His241(263) Nε2, Asp102(120) Oδ2 and the bridging water molecule, adopting a distorted tetrahedral geometry. However, in all B3 lactamases crystallized so far the coordination sphere in the Zn2 site adopts a trigonal bipyramidal geometry with two water ligands [START_REF] Ullah | The crystal strucuture of the L1 metallo-beta-lactamase from Stenotrophomonas maltophilia at 1.7 A resolution[END_REF][START_REF] Garcia-Saez | Three-dimensional structure of FEZ-1, a monomeric subclass B3 metallo-beta-lactamase from Fluoribacter gormanii, in native form and in complex with D-captopril[END_REF][START_REF] Docquier | High-resolution crystal structure of the subclass B3 metallo-betalactamase BJP-1: rational basis for substrate specificity and interaction with sulfonamides[END_REF][START_REF] Wachino | Structural insights into the subclass B3 metallo-beta-lactamase SMB-1 and the mode of on September 21, 2018 by guest http[END_REF][START_REF] Leiros | Crystal structure of the mobile metallo-beta-lactamase AIM-1 from Pseudomonas aeruginosa: insights into antibiotic binding and the role of Gln157[END_REF]. The presence of additional non-protein ligands in the GOB-18 site cannot be ruled out, given our data's resolution limit. In the same line of thought, an axial water molecule with high mobility was modeled in the coordination sphere of the Zn1 ion in both GOB-18 monomers, but a larger species (such as glycerol from the cryo-protection solution) cannot be excluded.

The conformation of the zinc ligands in GOB-18 active site is stabilized by a network of hydrogen bonds (Figure 4B), involving outer sphere ligands. His100(118) Nε2 and His175(196) Nδ1 interact respectively with Asp135(153) and Ser202(223) side chains, and Gln98(116) Nε2 establishes contacts with Asn199(220) Oδ1. Additionally, the Nδ1 atom of His103( 121) is Hbonded with the main chain N of residues Thr68(85) and Gly69(86) through a bridging water, His241(263) Nε2 interacts with the side chain of Asp51(67), and the carboxylate of Asp102(120) contacts its own amide nitrogen. Most of these outer sphere ligands are conserved in B3 enzymes [START_REF] Ullah | The crystal strucuture of the L1 metallo-beta-lactamase from Stenotrophomonas maltophilia at 1.7 A resolution[END_REF][START_REF] Garcia-Saez | Three-dimensional structure of FEZ-1, a monomeric subclass B3 metallo-beta-lactamase from Fluoribacter gormanii, in native form and in complex with D-captopril[END_REF][START_REF] Docquier | High-resolution crystal structure of the subclass B3 metallo-betalactamase BJP-1: rational basis for substrate specificity and interaction with sulfonamides[END_REF][START_REF] Wachino | Structural insights into the subclass B3 metallo-beta-lactamase SMB-1 and the mode of on September 21, 2018 by guest http[END_REF][START_REF] Leiros | Crystal structure of the mobile metallo-beta-lactamase AIM-1 from Pseudomonas aeruginosa: insights into antibiotic binding and the role of Gln157[END_REF], despite the divergence introduced by the His116Gln replacement.

A novel feature in GOB-18 can be observed at residue Cys180(201): its thiol and amide nitrogen make H-bonds with the backbone carbonyls of Leu96(114) and Thr97(115), respectively (Figure 3A). These residues are located in the β5-α3 loop, which contains the metal-binding motif Q 116 XH 118 XD 120 H 121 that replaces the HXHXDH signature present in the rest of B3 enzymes. Thus, Cys180(201), within the β-strand just downstream from the loop including on September 21, 2018 by guest http://aac.asm.org/ Downloaded from metal-binding His175(196), connects the bases of two loops that comprise five out of six metal ligands, and all the Zn1-coordinating residues.

The residues that define the substrate binding cleft in GOB-18 are mainly found in loops.

The catalytic pocket (Figure 5) is delimited by segments 23-32(30-39) (spanning a nonstructured coil and a short, kinked helix), [START_REF] Roe | PTRAJ and CPPTRAJ: software for processing and analysis of molecular synamics trajectory data[END_REF] and BJP-1. The wide substrate-binding groove in GOB enzymes correlates with high catalytic efficiencies for a broad spectrum of substrates, in contrast to BJP-1, in which an N-terminal helix partially covers the active site [START_REF] Docquier | High-resolution crystal structure of the subclass B3 metallo-betalactamase BJP-1: rational basis for substrate specificity and interaction with sulfonamides[END_REF].

Substrate docking and molecular dynamics simulations

We attempted to obtain a model for di-Zn(II) GOB-18 complexed with imipenem by in silico docking calculations. However, none of the resulting models reproduced binding modes consistent with a productive Michaelis complex. Similar docking simulations with L1 were instead successful, yielding a model which reproduced most of the binding features reported in on September 21, 2018 by guest http://aac.asm.org/ Downloaded from the crystal structure of L1 in complex with hydrolyzed moxalactam (PDB 2AIO) (52) (Figure S1).

In our docking model, the carbonyl C7 from imipenem is positioned close to the bridging OH - enabling the nucleophilic attack, and the carboxylate C9 interacts with the Zn2 ion, anchoring the substrate, in agreement with the generally accepted productive binding mode of β-lactam compounds to dinuclear MBLs. Based on these results, we generated a model of GOB-18 in complex with imipenem, by structural alignment of GOB-18 onto the L1-imipenem complex.

The L1-imipenem and GOB-18-imipenem models were used as starting geometries for molecular dynamics simulations, ran for 50 ns. In the L1-imipenem complex, residues Ser221 and Ser223 interact with the carboxylate C9 of imipenem throughout the simulation (Figure 6A). Additionally, the OH group C10 of imipenem established a hydrogen bond with the nonchelating oxygen of the Zn2-ligand Asp120. In GOB-18, Ser221 is replaced by a Met residue whose side chain points out of the active site, buried in the hydrophobic core of the protein, and hence unable to interact with the substrate (Figure 6B). This orientation is conserved throughout the dynamics. On the other hand, the imipenem carboxylate contacts residue Ser202(223) through a water molecule, and the interaction of the OH group C10 with Asp102(120) is also preserved in the GOB-18-imipenem complex. Taken together, these data strongly suggest that the Ser221Met substitution may have an impact on substrate binding by GOB enzymes.

The mobility of active site loops in MBLs has been related to substrate specificity. Indeed, residues 221 and 223 are located in a loop flanking the active site (Figure S2). We calculated the fluctuations of residues within this loop in GOB-18 and L1, both in the resting state and in the Michaelis complex with imipenem. The rmsf (root mean square fluctuation) of on September 21, 2018 by guest http://aac.asm.org/ Downloaded from this loop is substantially larger in GOB-18 than in L1 (Figure S2). We propose that the Ser221Met substitution present in GOB enzymes is compensated by a larger flexibility in this loop, which might assist substrate binding within the catalytic groove.

on September 21, 2018 by guest http://aac.asm.org/ Downloaded from

DISCUSSION

Here we report the crystal structure and biochemical characterization of the periplasmic di-Zn(II) form of GOB-18 from E. meningoseptica. The structural data reveal that a Gln residue replaces the ubiquitous His116 in the coordination sphere of the Zn1 ion (Figure 4). Periplasmic GOB-18 is a fully active broad spectrum lactamase in the di-Zn(II) form (Table 1), in agreement with a previous report that studied GOB-1 [START_REF] Horsfall | Broad antibiotic resistance profile of the subclass B3 metallo-beta-lactamase GOB-1, a di-zinc enzyme[END_REF]. These results contrast our previous study with recombinant GOB-18 obtained from the cytoplasm of E. coli cells [START_REF] Moran-Barrio | The metallo-beta-lactamase GOB Is a mono-Zn(II) enzyme with a novel active site[END_REF]. In that case, accumulation of the protein in the bacterial cytoplasm resulted in an inactive Fe(III)-bound form, which could be demetallated and subsequently loaded with Zn(II) in vitro to obtain an active mono-Zn(II) variant. Indeed, the preferential binding of a given divalent cation over others is influenced by the cellular localization of proteins [START_REF] Moran-Barrio | Secretion of GOB metallo-betalactamase in Escherichia coli depends strictly on the cooperation between the cytoplasmic DnaK chaperone system and the Sec machinery: completion of folding and Zn(II) ion acquisition occur in the bacterial periplasm[END_REF], and mismetallation upon protein overproduction in the cytoplasm of E. coli has been documented for a number of systems [START_REF] Tottey | Protein-folding location can regulate manganese-binding versus copper-or zinc-binding[END_REF][START_REF] Nar | product of heterologous expression in Escherichia coli of Pseudomonas aeruginosa copper azurin[END_REF][START_REF] Cotruvo | Metallation and mismetallation of iron and manganese proteins in vitro and in vivo: the class I ribonucleotide reductases as a case study[END_REF]. Thus, these separate preparations give us the opportunity to compare monoand di-Zn(II) variants of GOB-18.

We now show that when secreted into the bacterial periplasm, GOB exclusively binds Zn(II), which is relevant given that this is the physiological cellular compartment of MBLs in Gram-negative bacteria [START_REF] Moran-Barrio | Secretion of GOB metallo-betalactamase in Escherichia coli depends strictly on the cooperation between the cytoplasmic DnaK chaperone system and the Sec machinery: completion of folding and Zn(II) ion acquisition occur in the bacterial periplasm[END_REF]. Thus, in order to avoid chelation and remetallation steps, we optimized a protocol for the production of GOB-18 in the periplasm of E. coli and the preparation of high amounts of pure mature protein in the absence of affinity tags. Indeed, periplasmic GOB-18 preparations containing exclusively Zn(II) were successfully obtained and, while all efforts aimed at crystallizing the cytoplasmic Fe(III)-containing variant or the on September 21, 2018 by guest http://aac.asm.org/ Downloaded from reconstituted mono-Zn(II) form of GOB-18 were unsuccessful, diffraction quality crystals readily grew from solutions of periplasmic di-Zn(II) GOB-18. These results clearly show that the differences in metal content previously noted between GOB-1 and GOB-18 were due to the procedures employed to produce the recombinant proteins in each case and not due to the few residues differing in their primary structures. Taking all these data into account, it is safe to extrapolate several of the present conclusions to all GOB enzymes. Periplasmic di-Zn(II) GOB-18 catalyzed hydrolysis of β-lactam substrates with k cat and K M values similar to those reported for mono-Zn(II)-GOB-18 and di-Zn(II)-GOB-1 (Table 1). Besides, previous analyses of mutant GOB-18 Asp120Ser and metal-substituted GOB-18 derivatives, showed that the Zn2 site is essential for catalysis and for the stabilization of an anionic intermediate in the hydrolysis of nitrocefin [START_REF] Moran-Barrio | The metallo-beta-lactamase GOB Is a mono-Zn(II) enzyme with a novel active site[END_REF][START_REF] Lisa | Catalytic role of the metal ion in the metallo-beta-lactamase GOB[END_REF]. On the other hand, even though substitution of Gln116 by an isosteric His residue was somewhat detrimental for the resistance profile conferred by GOB-18 [START_REF] Moran-Barrio | The metallo-beta-lactamase GOB Is a mono-Zn(II) enzyme with a novel active site[END_REF][START_REF] Moran-Barrio | In vivo impact of Met221 substitution in GOB metallo-beta-lactamase[END_REF], it had little effect both on metal content as well as on the in vitro activity of dinuclear GOB-1 [START_REF] Horsfall | Broad antibiotic resistance profile of the subclass B3 metallo-beta-lactamase GOB-1, a di-zinc enzyme[END_REF] and remetallated mono-Zn(II) GOB-18 [START_REF] Moran-Barrio | The metallo-beta-lactamase GOB Is a mono-Zn(II) enzyme with a novel active site[END_REF]. Indeed, mutations Gln116Asn and Gln116Ala, which are predicted to impact on the structure of the Zn2 site through perturbations on the second coordination sphere (Figure 4B), reduced the catalytic efficiency of GOB-1 [START_REF] Horsfall | Broad antibiotic resistance profile of the subclass B3 metallo-beta-lactamase GOB-1, a di-zinc enzyme[END_REF]. Overall, the available data indicate that the Zn1 site in GOB enzymes contributes only marginally to activity, whereas it may be critical for the enzyme function in vivo. This would depend on the zinc availability, which can vary widely depending on environmental conditions [START_REF] Gonzalez | Metallobeta-lactamases withstand low Zn(II) conditions by tuning metal-ligand interactions[END_REF].

The finding of a dinuclear Zn(II) site where Gln116 acts as a metal ligand of the Zn1 ion contrasts with that found in B2 MBLs, where substitution of His116 by an Asn residue leads to a on September 21, 2018 by guest http://aac.asm.org/ Downloaded from dinuclear non-active species [START_REF] Fonseca | Crystal structure of Serratia fonticola Sfh-I: activation of the nucleophile in mono-zinc metallo-betalactamases[END_REF][START_REF] Garau | A metallobeta-lactamase enzyme in action: crystal structures of the monozinc carbapenemase CphA and its complex with biapenem[END_REF]. In the case of di-Zn(II)-CphA, residue Asn116 does not act as a metal ligand and the Zn1 ion is coordinated by only two protein residues, adopting a nonproductive position [START_REF] Bebrone | The structure of the dizinc subclass B2 metallobeta-lactamase CphA reveals that the second inhibitory zinc ion binds in the histidine site[END_REF]. Instead, the longer side chain of Gln116 (isosteric to a His residue) in GOB enzymes makes it a good metal ligand, so that the Zn1 ion adopts a position similar to that held in other di-Zn MBLs (Figure 7). The finding of a conserved network of hydrogen bonds among second sphere ligands confirms the requirement of a similar geometric arrangement in the active site. We have also found that a unique Cys residue may help anchoring the two loops that provide protein ligands for the Zn1 ion (Figure 3A). A conservative mutation of this Cys residue to a Ser (29) preserves the enzyme activity, supporting this hypothesis. The role of the Zn1 site in MBLs is to facilitate deprotonation of the bound water to provide an active nucleophile [START_REF] Meini | Overcoming differences: The catalytic mechanism of metallo-beta-lactamases[END_REF]. This function cannot be properly fulfilled by the Zn1 ion in B2 enzymes, thus resulting in enzyme inhibition [START_REF] Bebrone | The structure of the dizinc subclass B2 metallobeta-lactamase CphA reveals that the second inhibitory zinc ion binds in the histidine site[END_REF], but instead is fully preserved in GOB enzymes. Therefore, the His116Gln mutation is conservative in terms of both structure and function. The role of the Zn2 site is two-fold: to provide an anchoring electrostatic point for substrate binding and to stabilize the development of negative charge in the bridgehead nitrogen. Both functions also depend on the adequate positioning of this metal ion, which is indeed maintained in GOB-18.

Position 221 is essential in B1 and B2 MBLs, where a Cys residue acts as a ligand of the Zn2 ion. Instead, in most B3 enzymes a Ser is found in this position. The report of a Met residue in position 221 in GOB enzymes led us to speculate that the thioether moiety could act as a weak metal ligand. However, mutagenesis experiments in GOB-18 showed that Met221 is not involved in metal binding nor in catalysis, but that, instead, has a structural role [START_REF] Moran-Barrio | In vivo impact of Met221 substitution in GOB metallo-beta-lactamase[END_REF][START_REF] Lisa | Probing the Role of Met221 in the Unusual Metallo-beta-lactamase GOB-18[END_REF]. The crystal structure that we are now disclosing, confirms that Met221 is not a metal ligand, on September 21, 2018 by guest http://aac.asm.org/ Downloaded from actually its side chain does not point toward the substrate-binding site (Figure 6). Besides, docking and molecular dynamics simulations confirm that this residue is not involved in substrate binding, in sharp contrast to the role of the conserved Ser221 in most B3 enzymes.

Therefore, these results provide the first available evidence for an MBL where residue 221 is not involved in metal chelation nor in substrate binding, and highlight the diversity of roles fulfilled by this position among enzymes within the family. Additionally, a wide substratebinding groove in GOB-18 (Figure 5) correlates with a high catalytic efficiency against a diverse range of β-lactam substrates (Table 1) as compared to other B3 MBLs, in agreement with previous observations. The metal binding site of GOB enzymes is unusual not only among MBLs. Indeed, Asn/Gln ligands in protein zinc sites are very rare (0.5 % of the cases, according to Dudev and Lim) [START_REF] Dudev | First-second shell interactions in metal binding sites in proteins: a PDB survey and DFT/CDM calculations[END_REF]. Surprisingly, the His116Gln substitution does not have a direct impact on the broad spectrum profile of GOB-18. GOB is also peculiar in that its native organism, E. meningoseptica, is the only known bacterium expressing two MBLs: the B1 enzyme BlaB, and the B3 enzyme GOB. We have shown that, even if both genes are actively expressed, the higher levels of BlaB make this enzyme the one responsible for carbapenemase resistance [START_REF] Gonzalez | Carbapenem resistance in Elizabethkingia meningoseptica is mediated by metallo-beta-lactamase BlaB[END_REF]. Thus, GOB could be redundant in this organism despite its efficient catalytic performance. GOB-like alleles have also been found in metagenomics studies of a remote Alaskan soil with minimal human-induced selective pressure [START_REF] Allen | Functional metagenomics reveals diverse beta-lactamases in a remote Alaskan soil[END_REF]. More recently, new MBLs closely homologous to GOB enzymes have been found in Pedobacter roseus (PEDO-1), Pedobacter borealis (PEDO-2), Chrisobacterium piscium (CPS-1) and Epilithonimonas tenax (ESP-1) [START_REF] Gudeta | Biochemical Characterization of CPS-1, a Subclass B3 Metallo-beta-Lactamase from a Chryseobacterium piscium Soil Isolate[END_REF][START_REF] Gudeta | The Soil Microbiota Harbors a Diversity of Carbapenem-Hydrolyzing beta-Lactamases of Potential Clinical Relevance[END_REF]. All these MBLs feature the characteristic Gln116 residue in the active site, as well as the Cys residue in second sphere on September 21, 2018 by guest http://aac.asm.org/ Downloaded from position, and a hydrophobic residue in position 221 (Met or Leu), discussed in the present crystal structure, confirming that GOB-like alleles are ubiquitous in environmental bacteria. We can speculate that GOB genes could serve as a rather ancient resistance reservoir, or maybe could give rise to a different, yet unknown function, elicited by the presence of the Gln residue, providing at the same time an independent source of antibiotic resistance. In any case, these findings further highlight the large structural diversity of MBLs in current microorganisms, the details of which constitute valuable information in conceiving better antibiotic design strategies.

on September 21, 2018 by guest http://aac.asm.org/ on September 21, 2018 by guest

Downloaded from

  and the model of native FEZ-1 from Legionella gormanii (PDB 1K07) as search probe. The asymmetric unit contains two monomers of GOB-18 (chains A and B) with ca. 44% of the volume occupied by solvent. SigmaA-weighted on September 21, 2018 by guest http://aac.asm.org/ Downloaded from Fourier maps (2mFo-DFc) allowed rebuilding the initial model reliably through iterative cycles of manual model building with COOT (37) and refinement with BUSTER (38). To minimize modelbias, initial refinement cycles were carried out including high-temperature (7,500K) slowcooling simulated annealing procedures in torsional space (as implemented in the program PHENIX (39)). The final model revealed two outlier residues in the Ramachandran plot. Figures were generated and rendered with Pymol 1.5.0.2. (Schrödinger, LLC). Volume calculations for automatically identified cavities were done using the CASTp algorithm (40) and Q-site finder (41), giving similar results. Atomic coordinates and structure factors were deposited in the Protein Data Bank under the accession code 5K0W. Substrate docking and molecular dynamics simulations. Molecular docking was performed using Autodock 4.2 (42). Deprotonated imipenem was docked setting up a docking box of 40 × 40 × 40 Å (grid-point spacing of 0.375 Å) centered in the enzyme active site. The model of GOB-

  -52(67-68), 98-103(116-121) (including the QXHXDH motif), 135-144(154-163) (encompassing a short helix), 175-176(196-197) (containing metalbinding His175(196)), 199-204(220-225) (harboring residue Met 200(221)) and 240-244(262-266) (spanning a short helix and including metal-binding His241(263)). This cleft, albeit shallow, is one of the top ranking cavities, comprising 150-200 Å 3 with approximate 8 Å x 15 Å on the opening and 10 Å deep. A second, smaller pocket, with residues Ser202(223), Gln244(266), Asn271(293) and Leu275(297) conforming the side limits and Met200(221) and Trp237(259) on the floor, is immediately adjacent to the first one, thus constituting a discontinuous groove.

Figure 5

 5 Figure 5 compares the catalytic groove of GOB-18 with those from the B3 lactamases FEZ-1, L1

FIGURE 1 .

 1 FIGURE 1. Metallo-beta-lactamase zinc-binding sites. B. cereus BcII (B1, PDB 1BC2, left), S. fonticola SfhI (B2, PDB 3SD9, center), and S. maltophilia L1 (B3, PDB 1SML, right). Zinc atoms are shown as gray spheres, and water molecules (W) are shown as small red spheres. Coordination bonds are indicated with dashed lines.

FIGURE 2 .

 2 FIGURE 2. Purification and characterization of periplasmic GOB-18. (A) SDS-PAGE analysis of fractions along different steps of the purification process. Lanes: 1, total cell extract (T); 2, periplasmic fraction, corresponding to the sample loaded on the CM-Sephadex column (L); 3, CM-Sephadex flow-through (F CM ); 4, CM-Sephadex elution, corresponding to the sample loaded on the Mono S column (E); 5, Mono S flow through (F S ); 6-17, Mono S elution fractions (f1 to f12). Molecular weight markers (in kDa) are indicated at the right side. The vertical grey line between lanes 3 and 4 indicates the junction of two different gels prepared and run together, since the Mini PROTEAN III System (BioRad) holds up to 15 lanes. (B) Circular dichroism spectra of periplasmic GOB-18 (black line), remetallated Zn(II)-GOB-18 (gray) and apo-GOB-18 (light gray).

FiIGURE 3 .

 3 Overall structure of periplasmic GOB-18. (A) Cartoon representation of GOB-18 (chain A) with helices colored in red, strands in yellow and non-structured loops in green. The inset highlights selected residues (in stick representation). Dashed lines represent atomic interactions. (B) Comparison of GOB-18 with the B3 MBLs FEZ-1 (PDB 1K07), BJP-1 (PDB 3LVZ), on September 21, 2018 by guest http://aac.asm.org/ Downloaded from and L1 (PDB 1SML). The segments of GOB-18 exhibiting the highest rmsd values are highlighted in colors. Zn atoms are shown as gray spheres.

FIGURE 4 .

 4 FIGURE 4. The active site of periplasmic GOB-18. (A) Upper panel: Comparison of the active site in the two monomers present in the crystal structure of GOB-18. Chain A is colored by atom in yellow (C atoms), blue (N atoms), red (oxygen atoms) and dark gray (zinc atoms). Chain B is shown in light grey throughout. Protein residues are depicted in sticks, zinc atoms are shown as big spheres and water molecules as small spheres. The 2mFo-DFc sigmaA-weighted electron density, contoured at 1.7 σ and represented as a grey mesh, corresponds to chain A. Dashed lines represent atomic interactions and the related distances are of 2.1 Å in all cases except for the interactions Zn1-Gln98 and Zn1-axial water where distances are 2.0 Å and 2.2 Å, respectively. Lower panel: comparison of the active site of GOB-18 (chain A, yellow) with those of the B3 MBLs FEZ-1 (PDB 1K07, pink), BJP-1 (PDB 3LVZ, green), and L1 (PDB 1SML, blue). (B) Residues in the second coordination sphere in the active of GOB-18 (chain A) are shown in stick representation with C atoms colored in cyan.

FIGURE 5 .FIGURE 6 .

 56 FIGURE 5. The catalytic groove in GOB-18. Surface representation of GOB-18 (chain A) highlighting with colors the structural elements that define the catalytic groove. Zinc atoms are shown as gray spheres. For comparison, the region delimited by the rectangle is compared to the equivalent regions of the B3 MBLs FEZ-1 (PDB 1K07), L1 (PDB 1SML) and BJP-1 (PDB 3LVZ) shown at the bottom.

FIGURE 7 .

 7 FIGURE 7. Comparison of the active sites of di-Zn(II) GOB-18 and di-Zn(II) CphA. GOB-18 (chain A) is shown in light gray and CphA (PDB 3F9O) is colored in orange (C atoms), blue (N atoms), red (oxygen atoms) and dark gray (zinc atoms). Protein residues are shown in sticks, zinc atoms are depicted as spheres and dashed lines highlight atomic interactions.

  multiplicity for each reflection; I i , the intensity of the i th observation of reflection h; <I>, the mean of the intensity of all observations of reflection h, ); ∑ is taken over all reflections; ∑ is taken over all observations of each reflection. ‡= ∑ | (ℎ) -(ℎ) |/ ∑ | (ℎ)| ; R cryst and R free were calculated using the working and test hkl reflection sets, respectively. ‡ ‡ Calculated with Phenix get_cc_mtz_pdb (39). § Calculated with Mol Probity (62).

Table 1 .

 1 Kinetic parameters of hydrolysis of β-lactam substrates by GOB enzymes. Values were derived from a nonlinear fit of Michaelis-Menten equation to initial rate measurements and correspond to the average of at least three independent enzyme preparations. k cat values are expressed in s -1 , K M in µM and k cat /K M in (s -1 µM).

	TABLES
	Substrates Imipenem Meropenem	Zn(II) added 0 μM 20 μM 0 μM 20 μM	Periplasmic GOB-18 k cat K M k cat /K M 37 ± 1 22 ± 3 1.7 ± 0.5 75 ± 3 42 ± 7 1.8 ± 0.5 96 ± 4 54.3 ± 6 1.8 ± 0.6 261 ± 9 170 ± 20 1.5 ± 0.7	Cytoplasmatic GOB-18 k cat K M k cat /K M a 42 ± 9 26 ± 2 1.6 ± 1.5 72 ± 0.5 40 ± 10 1.8 ± 0.5	added 0 μM 50 μM 0 μM 50 μM Zn(II)	k cat 77 ± 2 85 ± 2 100 ± 7 170 ± 3	K M 18 ± 0.6 13 ± 1 29 ± 1 22 ± 1 GOB-1 b	k cat /K M 4.2 6.5 3.5 8.0	http://aac.asm.org/ Downloaded from
	Cefaloridine Cefotaxime Penicillin-G	0 μM 20 μM 0 μM 20 μM 0 μM 20 μM	41 ± 2 59 ± 2 80 ± 3 106 ± 5 130 ± 20 0.8 ± 0.3 100± 10 0.4 ± 0.1 150 ± 20 0.4 ± 0.1 100 ± 10 0.8 ± 0.3 1070 ± 20 450 ± 20 2.4 ± 0.9 1810 ± 40 580 ± 50 3.1 ± 0.9	30 ± 2 83 ± 2 680 ± 80 330 ± 30 2.1 ± 0.4 31 ± 0.5 0.95 ± 0.08 88 ± 6 0.94 ± 0.09	0 μM 50 μM 630 ± 10 190 ± 10 540 ± 7 130 ± 6	4.2 3.4	on September 21, 2018 by guest

a Values correspond to mono-Zn(II)-GOB in reference

[START_REF] Moran-Barrio | The metallo-beta-lactamase GOB Is a mono-Zn(II) enzyme with a novel active site[END_REF]

.

b Values correspond to reference

[START_REF] Horsfall | Broad antibiotic resistance profile of the subclass B3 metallo-beta-lactamase GOB-1, a di-zinc enzyme[END_REF]

.

Table 2 .

 2 X ray diffraction data collection and refinement statistics.

	701
	702
		GOB-18
	Space group	P2 1
	Protein molecules per asymmetric unit Solvent content (%) Wavelength (Å) Data Resolution (Å) * Measured reflections Multiplicity * Completeness (%) * R meas (%) * † <I/σ(I)> * a b c (Å) ß (°) Refinement resolution (Å) R cryst ‡ (N° refs) R free ‡ (N° refs) Rms bonds (Å) Rms angles (degrees) Protein non-hydrogen atoms Water atoms Zn +2 atoms Glycerol atoms	2 43.9 1.5418 24.33-2.61 (2.75-2.61) 2.8 (2.6) 96.9 (90.5) 15.1 (45.3) 9.5 (3.3) 64.0 48.5 88.9 24.3-2.61 0.192 (15152) 0.242 (955) 0.01 1.11 4381 77 4 6 100.1 44417	on September 21, 2018 by guest http://aac.asm.org/ Downloaded from
	Cl -1 atoms	8
	Mean B factor -overall : [chain A / chain B] (Å 2 )	28 / 31
	Mean B factor -main chain: (chain A / chain B) (Å 2 )	25 / 27

* Values in parentheses apply to the high-resolution shell.

ACKNOWLEDGEMENTS

We thank Dr. R. Girolami for atomic absorption measurements. MNL is a postdoctoral fellow from ANII and was a recipient of a doctoral fellowship from CONICET. SID is recipient of a doctoral fellowship from CONICET. JMB, DMM, AMV and AJV are staff members from CONICET. This work was supported by grants from ANPCyT and the US National Institutes of Health (1R01AI100560) to AJV; from ANPCyT, CONICET, UNR, and Ministerio de Salud, Provincia de Santa Fe, Argentina to AMV; from ANPCyT and CONICET to DMM.


During a study on the prevalence and diversity of campylobacteria in wild birds faecal samples in the city of Valdivia (South of Chile) 17 Gram-negative, curved rod isolates, were initially identified as Campylobacter lari by PCR-RFLP. Further identification by 16S rRNA sequence analysis revealed that they formed a distinct group in the genus Campylobacter. This unique position was confirmed by rpoB, atpA and cpn60 gene sequence analysis. The average nucleotide identity between the representative strain WBE38 T and the type strain of the closest taxon Campylobacter lari subsp.

 (LMG 11760)was 90.8%. The oxidase and urease activity of the novel isolates enabled them to be phenotypically differentiated from recognized Campylobacter species.

Therefore, on the basis of phenotypic, genetic and genomic characterizations, this study clearly shows that these strains represent a novel species within the genus Campylobacter, for which the name Campylobacter ornithocola sp. nov. is proposed, with the type strain WBE38 T (=CECT 9147 T =LMG 29815 T ).

3

The genus Campylobacter was proposed by [START_REF] Sebald | Base DNA content and classification of vibrios[END_REF], to accommodate the species Vibrio fetus (now Campylobacter fetus, the type species of the genus). Since then the number of Campylobacter species has greatly increased and, at the time of writing [START_REF] Korczak | Genetic relatedness within the genus Campylobacter inferred from rpoB sequences[END_REF], this genus encompassed 28 species and 9 subspecies with valid published names (LPSN, 2016;[START_REF] Piccirillo | Campylobacter geochelonis sp. nov. isolated from the western Hermann's tortoise (Testudo hermanni hermanni)[END_REF][START_REF] Van | Campylobacter hepaticus sp. nov., isolated from chickens with spotty liver disease[END_REF]. The Campylobacter infection is the leading cause of foodborne bacterial gastroenteritis in the developed world, where infection is principally the result of consumption of contaminated meat and poultry.

The most common symptoms of campylobacteriosis include severe diarrhoea, with a proportion of patients developing chronic sequelae such as Guillain-Barré syndrome, reactive arthritis and irritable bowel syndrome [START_REF] Man | The clinical importance of emerging Campylobacter species[END_REF]. Campylobacter jejuni and Campylobacter coli are by far the most isolated and studied species, while the epidemiology and the clinical role of the other less common species in human and animal diseases is far less understood [START_REF] Man | The clinical importance of emerging Campylobacter species[END_REF][START_REF] Iraola | Genomic evidence for the emergence and evolution of pathogenicity and niche preferences in the genus Campylobacter[END_REF].

In the present study, 17 new Campylobacter isolates recovered from wild bird faecal samples were subjected to a polyphasic approach including molecular identification by PCR-RFLP, genotyping by ERIC-PCR, phylogenetic analysis using the 16S rRNA, rpoB, atpA and cpn60 genes and phenotypic characterization, in order to determine their taxonomic position. Additionally, the whole genome of WBE38 T was sequenced, since it was selected as the type strain. Based on our results, we propose and describe these strains as a novel species within the genus Campylobacter.

Over the period between January 2013 and October 2015, a study was conducted to evaluate the prevalence and diversity of campylobacteria in wild bird faecal samples in the city of Valdivia, South of Chile, where a collection of Campylobacter isolates was obtained 4 (L. Collado, paper in preparation). The samples corresponded to bird's faecal droppings excreted in public urban parks. Although the specific type of bird corresponding to each specimen remains unidentified, at the time of sampling black-faced ibis (Theristicus melanopis), southern lapwing (Vanellus chilensis) and chimango caracara (Milvago chimango) were the main species observed at these sites. Faecal samples were collected using sterile cotton-tipped swabs that were immediately placed into 8 ml Bolton broth (Oxoid). Incubation was performed at 37 °C for 48 h under micro-aerobic conditions (CampyGen, Oxoid). After enrichment, an aliquot (400 µl) was transferred onto the surface of a 0.45 µm membrane filter (Millipore) which was placed on a Petri dish containing Blood Agar Base (Merck) supplemented with 5% defibrinated sheep blood (Quad Five) and it was allowed to filter passively under room conditions for 30 min. After filtration, the filters were removed with sterile forceps and discarded. The inoculated plates were incubated under the aforementioned conditions [START_REF] Collado | Description of Helicobacter valdiviensis sp. nov., an Epsilonproteobacteria isolated from wild bird faecal samples[END_REF]. The 17 novel isolates described here were initially identified as Campylobacter lari by the PCR-RFLP described in [START_REF] Marshall | Rapid identification of Campylobacter, Arcobacter, and Helicobacter isolates by PCR-restriction fragment length polymorphism analysis of the 16S rRNA gene[END_REF] showing the C1 and C1b restriction patterns with the endonucleases DdeI and BsrI, respectively (data not shown). However, recently this species was divided into two subspecies (C. lari subsp. lari and C. lari subsp. concheus) (Debuyne et al., 2009). Additionally, several C. lari-like new species were described, i.e., C. insulaenigrae [START_REF] Foster | Campylobacter insulaenigrae sp. nov., isolated from marine mammals[END_REF], C. peloridis (Debuyne et al., 2009), C. subantarticus (Debuyne et al., 2010a) andC. volucris (Debuyne et al., 2010b), that collectively have been referred to as Campylobacter lari group (Miller et al., 2014b). Since no available PCR method could differentiate these highly related taxa, all isolates we identified as C. lari by PCR-RFLP were further analyzed by a polyphasic taxonomic approach. Downloaded from www.microbiologyresearch.org by IP: 157.99.52.18 On: Fri,07 Apr 2017 14:41:25 5 The 17 isolates were first analysed by means of the enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) technique using the [START_REF] Houf | Assessment of the genetic diversity among arcobacters isolated from poultry products by using two PCR-based typing methods[END_REF] protocol, in order to avoid further analysis of strains with the same genotype. The electrophoretic patterns were analysed using the Jaccard index and a dendrogram was constructed by using the unweighted pair group linkage analysis (UPGMA) method with the Phoretix 1d Pro software (Totallab). As shown in Fig. 1, all the isolates belonged to different ERIC-PCR types and, consequently, were considered different strains. A nearly complete 16S rRNA gene sequence (1350 bp) of five selected strains (WBE38 T , WBE186, WBE206, WBE215 and WBE241), were amplified as described by [START_REF] Brenner | Classification of Centers for Disease Control group eugonic fermenter (EF)-4a and EF-4b as Neisseria animaloris sp. nov.and Neisseria zoodegmatis sp. nov., respectively[END_REF]. Additionally, the rpoB, atpA and cpn60 genes were also evaluated with the protocols described by [START_REF] Korczak | Genetic relatedness within the genus Campylobacter inferred from rpoB sequences[END_REF]), Miller et al. (2014a) and [START_REF] Hill | Identification of Campylobacter spp. and discrimination from Helicobacter and Arcobacter spp. by direct sequencing of Downloaded from www[END_REF], respectively. Due to the high number of strains, only the analysis of rpoB gene sequences included all 17 strains. Both DNA strands of the PCR products were directly sequenced with an ABI 3730 XL automatic DNA sequencer (ABI) by a commercial sequencing facility (Macrogen, Seoul, Korea).

Alignment of the sequences was performed with the CLUSTAL W program [START_REF] Thompson | CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, Downloaded from www[END_REF]. Phylogenetic trees were reconstructed with the MEGA 6 software [START_REF] Tamura | MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[END_REF], by using the neighbour-joining method [START_REF] Saitou | The neighbor-joining method: a new method for reconstructing phylogenetic trees[END_REF]) with Kimura's twoparameter substitution model [START_REF] Kimura | A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences[END_REF] and the stability of the groupings, estimated by bootstrap analysis (500 replications). Similarity values between the 16S rRNA gene sequence of strain WBE38 T and the type strains of all members of the genus Campylobacter with valid published names were calculated with the EzTaxon-e server [START_REF] Kim | Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species[END_REF] obtaining a similarity range of 99.5-90.7%. The 16S rDNA phylogenetic tree (Fig. 2 For the whole genome sequencing of strain WBE38 T , genomic libraries were prepared with the Nextera® XT DNA Sample Perparation Kit (Illumina, Inc.) and then sequenced using a MiSeq Illumina platform which produced 5,250,728 pair-end reads (2x150 cycles). After initial quality check reads were assembled with SPAdes [START_REF] Bankevich | SPAdes: a new assembly algorithm and its applications to single-cell sequencing[END_REF] and annotated with Prokka [START_REF] Seemann | Prokka: rapid prokaryotic genome annotation[END_REF], producing 160 contigs that were deposited in the GenBank under the accession number LXSU00000000. The version described in this paper is version LXSU01000000. The average nucleotide identity (ANI) was used as an alternative to DNA-DNA hybridization (DDH) [START_REF] Konstantinidis | Genomic insights that advance the species definition for prokaryotes[END_REF]. The ANIb (based on BLAST) values of WBE38 T compared to the sequenced type strains of all other species of the C. lari group were calculated using JSpecies v1.2.1 (Richter & Rosselló-Mora, 2009) and were below the 95% species cut-off (Table 1) [START_REF] Konstantinidis | Genomic insights that advance the species definition for prokaryotes[END_REF]. The genomic G+C content of WBE38 T was calculated using in-house R scripts and resulted in 29.5%, which is within the range reported for the genus Campylobacter (29-47%) [START_REF] Debruyne | Taxonomy of the family Campylobacteraceae[END_REF].

For the physiological and biochemical characterizations of all the new strains, phenotypic testing was performed as described previously (On & Holmes, 1991a[START_REF] On | Assessment of enzyme detection tests useful in identification of campylobacteria[END_REF]Ursing et al., 1995). Growth of strains was determined on nutrient broth no. 2 (Oxoid) supplemented with 5% defibrinated sheep blood (Quad Five) and 2% agar (Merck). Micro-aerobic growth (using CampyGen, Oxoid) was evaluated at 25°C, 37°C and 42°C for 48 to 72h. Aerobic and anaerobic growths (using AnaeroGen, Oxoid) were evaluated at 37°C for 72h.

7

Catalase activity was evaluated by adding a 3% H2O2 solution and observing the reaction within 5s. Oxidase activity was determined with Bactident Oxidase strips (Merck). Indoxyl acetate hydrolysis was determined as described by [START_REF] Mills | Hydrolysis of indoxyl acetate by Campylobacter species[END_REF]. In addition to this, a set of additional phenotypic tests (reduction of nitrates, esterase activity, hydrolysis of hippurate, γ-glutamyl transferase activity, reduction of triphenyl-tetrazolium chloride (TTC), alkaline phosphatase activity, production of H2S, assimilation of glucose and pyrrolidonyl-, L-arginine-and L-aspartate-arylamidase activities) were evaluated by using the API Campy identification system (bioMérieux) according to the manufacturer's showae [START_REF] Vandamme | Genus I. Campylobacter Sebald and Véron[END_REF]. However, the new isolates could be differentiated from oxidase negative campylobacters by the urease test. Table 2 shows the most important phenotypic characteristics differentiating the novel strains from the other species of the genus Campylobacter. Bacterial cell shape was observed using a scanning electron microscope (Zeiss, LEO 420) following the protocol described by [START_REF] Kawamura | Porphyromonas pogonae sp. nov., an anaerobic but low concentration oxygen adapted coccobacillus isolated from lizards (Pogona vitticeps) or human clinical specimens, and emended description of the genus Porphyromonas Shah and Collins 1988[END_REF].

The organism exhibits a curved shape and cells became spherical or coccoid after 72h of incubation (Fig. 3).

In conclusion, the results from this taxonomic study clearly demonstrate that the isolates recovered from wild birds faecal samples comprise a novel species distinct from other currently known Campylobacter species, based on 16S rRNA, housekeeping genes, whole Cells are Gram-negative curved rods, non-encapsulated, non-spore-forming and are 0.3-0.5 µm wide and 1.2-3 µm long. After incubation on Colombia agar (5% sheep blood) in microaerobic atmosphere at 37 °C for 48 h, colonies are glossy, slightly convex, round with smooth margins. Coccoid cells were observed in old cultures. Swarming on solid media was noted. Pigments are not produced. Grows on blood agar at 37 °C and at 42 °C under micro-aerobic culture conditions (does not require atmospheric hydrogen) and at 37°C in anaerobic conditions. No growth was observed at 37 °C on aerobiosis and at 25 °C under micro-aerobic conditions. No haemolysis is seen on blood agar. Catalase-and ureaseactivity is present but no oxidase. Esterase activity and reduction of Triphenyltetrazolium chloride (TTC) are variable. Pathogenicity is unknown; strains have been recovered from faecal samples of wild birds.

The type strain is WBE38 T (=CECT 9147 T = LMG 29815 T ) which was isolated from a faecal sample in Valdivia, Chile. [START_REF] Foster | Campylobacter insulaenigrae sp. nov., isolated from marine mammals[END_REF][START_REF] Matsuda | Urease-Positive Thermophilic Campylobacter species[END_REF][START_REF] Debruyne | Taxonomy of the family Campylobacteraceae[END_REF][START_REF] Debruyne | Novel Campylobacter lari-like bacteria from humans and molluscs: description of Campylobacter peloridis sp. nov., Campylobacter lari subsp. concheus subsp. nov. and Campylobacter lari subsp. lari subsp. nov[END_REF]2010a,b;[START_REF] Piccirillo | Campylobacter geochelonis sp. nov. isolated from the western Hermann's tortoise (Testudo hermanni hermanni)[END_REF][START_REF] Van | Campylobacter hepaticus sp. nov., isolated from chickens with spotty liver disease[END_REF]

  ) clearly indicated that the five strains represented a single species within the genus Campylobacter, most closely related to C. subantarticus (99.5% similarity) and C. lari subsp. concheus (99.4% similarity). The taxonomic position of the new strains within Downloaded from www.microbiologyresearch.org by IP: 157.99.52.18 On: Fri, 07 Apr 2017 14:41:25 6 the Campylobacter lari group and the demonstration that they represent a novel lineage were confirmed by the rpoB, atpA and cpn60 phylogenetic trees (Supplementary Fig. 1, 2 and 3, respectively).

  instructions. All tests were performed at least twice with C. jejuni (DSM 4688 T ), C. coli (DSM 4689 T ), C. lari (DSM 11375 T ), C. subantarticus (LMG 24377 T ), C. insulaenigrae (LMG 22716), C. volucris (LMG 24380 T ) and Escherichia coli (ATCC 25922) used as controls. The novel isolates were biochemically different from most of the recognized species of the genus Campylobacter because oxidase activity was not detected. This test is positive in all species except C. gracilis and sporadic isolates of C. concisus and C.

  Downloaded from www.microbiologyresearch.org by IP: 157.99.52.18 On: Fri, 07 Apr 2017 14:41:25 8 genome sequence comparison, morphological, physiological and biochemical properties. The name Campylobacter ornithocola sp. nov. is proposed, with WBE38 T (=CECT 9147 T = LMG 29815 T ) as the type strain. Description of Campylobacter ornithocola sp. nov. Campylobacter ornithocola [or.ni.tho'co.la. Gr. n. ornis, -ithos bird; L. suff. -cola (from L. n. incola) dweller; N.L. n. ornithocola bird dweller].

Fig. 2 .

 2 Fig. 2. Neighbour-joining tree based on 16S rRNA sequences showing the phylogentic

Fig. 3 .

 3 Fig. 3. Images of cells of strain WBE38T as observed with scanning electron microscopy.

Figure 1

 1 Figure 1 Click here to download Figure Fig. 1 (ERIC-PCR).pptx

Figure 2

 2 Figure 2 Click here to download Figure Fig. 2 (16S rRNA).pptx

  Figure 3 Click here to download Figure Fig. 3 (SEM).pptx

Table 1 .

 1 Average nucleotide identity based on BLAST (ANIb) values (in percentages) for C. ornithocola sp. nov. and the most closely related members of the genus Campylobacter Strains: 1, C. ornithocola sp. nov. WBE38 T ; 2, C. lari subsp. concheus LMG 11760; 3, C. lari subsp. lari RM 2100; 4, C. subantarticus LMG 24377 T ; 5, C. peloridis LMG 23910 T ; 6, C. volucris LMG 24379; 7, C. insulaenigrae NCTC 12927

	Strains 1	2	3	4	5	6	7
	1	-
	2	90,8	-
	3	89,5 92,8	-
	4	88,4 89,7 88,8	-
	5	86,0 86,2 86,1 85,3	-
	6	82,0 82,3 83,1 81,8 82,4	-
	7	80,9 81,0 81,2 80,2 81,1 82,9 -

Table 2 .

 2 Phenotypic characteristics that differentiate Campylobacter ornithocola sp. nov. from other Campylobacter species.

	336
	337 338 339 340	Campylobacter species: 1, C. ornithocola sp. nov (n = 17); 2, C. avium; 3, C. canadensis; 4, C. coli; 5, C. concisus; 6, C. corcagiensis; 7, C. cuniculorum; 8, C. curvus; 9, C. fetus subsp. fetus; 10, C.fetus subsp. testudinum; 11, C. fetus subsp. venerealis; 12, C. geochelonis; 13, C. gracilis; 14, C. helveticus; 15, C. hepaticus; 16, C. hominis; 17, C. hyointestinalis subsp. hyointestinalis; 18, C. hyointestinalis subsp. lawsonii; 19. C. iguanorum; 20, C. insulaenigrae; 21, C. jejuni subsp. doylei; 22, C. jejuni subsp. jejuni; 23, C. lanienae; 24, C. lari subsp. concheus; 25, C. lari subsp. lari; 26, C. mucosalis; 27, C. peloridis; 28, C. rectus; 29, C. showae; 30, C. sputorum; 31, C. subantarticus; 32, C. upsaliensis; 33, C. ureolyticus;
		34, C. volucris. Data for reference taxa were taken from

  Oxidase negative test allows differentiating the new species from Urease-Positive Thermophilic Campylobacter (UPTC) strains. † This test was determined for the corresponding type strain, in this study ‡ We obtained a different result from that published byDebruyne et al. (2010b), when we evaluated the nitrate test with the API Campy (nitrate reduction negative for strain LMG Cluster analysis based on the patterns obtained by ERIC-PCR from the Campylobacter ornithocola sp. nov. strains.

		Figures legends
		Fig. 1.
	341 342	100% strains positive; -, 100% strains negative; (+), 80-94% strains positive; V, 42-66% strains positive; (-), 7-33% strains positive; ND, not determined.						. +,
	Characteristic (mol %)	1 29.5	2 35	3 ND	4 31	5 37-41	6 31.9	7 32.4	8 45-46	9 33-35	10 ND	11 33-34	12 33.6	13 44-46	14 34	15 27.9	16 32.5	17 35-36	18 31-33	19 36	20 ND	21 31	22 30-31	23 36	24 30	25 29-30	26 36-38	27 29	28 45-46	29 44-46	30 29-33	31 30	32 32-36	33 28-30	34 29

*

Acknowledgements

We thank Bernhard Schink (University of Konstanz, Konstanz, Germany) for his help with the specific etymology and nomenclature. We also thank Ricardo Silva (Universidad Austral de Chile) for his help with SEM. Downloaded from www.microbiologyresearch.org by IP: 157.99.52.18 On: Fri, 07 Apr 2017 14:41:25

Funding information

This work was supported by the Comisión Nacional de Investigación Científica y Tecnológica (CONICYT-Chile) under the project FONDECYT Nº 11130402.

Conflicts of interest

The authors have no conflict of interest to declare.

Ethical statement

No experiments with humans or animals were carried out.

Reduction of

Nitrate

Hydrolysis of

Hippurate

Downloaded from www.microbiologyresearch.org by IP: 157.99.52.18

On: Fri, 07 Apr 2017 14:41:25


S Vahid Amirinezhad
email: vahid.amirinezhad@msor.vuw.ac.nz

Peter Donelan
email: peter.donelan@vuw.ac.nz

Kinematic Singularities of a 3-DoF Planar Geared Robot Manipulator

Keywords: planar manipulator, kinematic singularity, geared mechanism, matroid method

. We use this relatively simple mechanism to illustrate singularity analysis for geared mechanisms.

Introduction

The use of gear pairs in a mechanism may confer a number of advantages. For example, they can enable more efficient placement of the actuators thereby reducing their mass and inertia. Epicyclic gear trains (EGTs), in which the centre of one gear wheel revolves around that of another, are the simplest form and therefore play an important role in geared mechanisms (GMs). By utilising EGTs, we can easily place actuators close to the base of a GM and rotation of inputs can be efficiently transmitted to the end-effector. Careful choice of gear ratios can also enable endeffector motion to be tailored to specific inputs.

The fundamental kinematic equation for an epicyclic gear is due to Willis [START_REF] Willis | Principles of Mechanism[END_REF]. Subsequent authors have introduced methods of global analysis for GMs that ensure the equations are correctly formulated for a given mechanism topology and design. Notably, Buchsbaum and Freudenstein [START_REF] Buchsbaum | Synthesis of kinematic structure of geared kinematic chains and other mechanisms[END_REF] introduced combinatoric meth-ods to represent the topology of the mechanism. This approach was later developed by Tsai [START_REF] Tsai | The kinematics of spatial robotic bevel-gear trains[END_REF], Hsu and Lam [START_REF] Hsu | A new graph representation for the automatic kinematic analysis of planetary spur-gear trains[END_REF]. In order to enhance the computational effectiveness of the method, Talpasanu et al. [START_REF] Talpasanu | Kinematics and Dynamics of Mechanical Systems Based on Graph-Matroid Theory[END_REF][START_REF] Talpasanu | Application of matroid method in kinematic analysis of parallel axes epicyclic gear trains[END_REF] refined and to some extent recast the approach, introducing the 'incidence and transfer method' that uses the cycle matroid of the mechanism's directed graph. A comparison of Talpasanu's method with that of Tsai-Tokad was made in [START_REF] Amirinezhad | Kinematic analysis of geared robotic mechanism using matroid and T-T graph methods[END_REF]. In this paper, we illustrate Talpasanu's method for a simple geared version of a planar 3R mechanism in order to determine its kinematic mapping and thereby its singularities. The goal is to develop a systematic approach to the kinematic analysis of GMs and the determination of their singularities. While the example presented is straightforward, it is intended to provide a model for extending singularity analysis of GMs to more complex cases, including those which are genuinely spatial in their kinematics and to parallel mechanisms incorporating gearing.

The Mechanism

A simple planar GM consists of n + 1 links, L 0 , . . . , L n , and m joints that include t revolute (turning) pairs, T 1 , . . . , T t , and g gear pairs, G 1 , . . . , G g , so that m = t + g. Note that the number of links, excluding the base L 0 , is assumed equal to the number of simple pairs, i.e. t = n. In effect, the mechanism without gears contains no closed chains.

By placing three actuator joints at the base and using simple spur-gear pairs to transmit motion to the end-effector, one obtains a geared mechanism based on a simple serial planar 3R mechanism (see Figure 1). One EGT, consisting of three gear wheels and using link L 1 as carrier, transmits motion to the link L 7 , while a second EGT of five gear wheels with links L 1 and L 7 as carriers transmits motion to the end-effector. A functional schematic for the mechanism is illustrated in Figure 2. The inputs, which are attached to the base L 0 , are via T i , i = 1, 2, 3 while link L 9 is the output planet gear or end-effector. Note that the carrier arms L 1 and L 7 that form the first two links in the underlying planar 3R are also gear wheels. Other links are intermediate (idler) gear wheels.

G 2 L 1 L 2 L 3 L 4 L 5 L 6 L 7 L 8 L 9 T 2 T 1 T 3 T 4 T 5 T 7 T 6 T 8 T 9 G 1 G 3 G 4 G 5 G 6 y 0 z 0 O r 3 r 5 r 7 r 4 r 6 r 8 r 9 r 1 r 2 l 1 l 7
Fig. 2 Functional schematic of manipulator in Figure 1 In its directed graph (digraph) representation, Figure 3, the links (including gear wheels) are vertices (L 0 , . . . , L 9 ) while joints are edges. Specifically, the revolute pairs (T 1 , . . . , T 9 ) are solid edges and gear pairs (G 1 , . . . , G 6 ) are dashed. Note that the solid edges form a spanning tree for the graph; put another way, each simple cycle contains at least one gear pair as an edge. The direction of an edge connecting vertices (links) L i and L j is L i → L j if the transmission from input to output flows from L i to L j .

Application of the CGK formula shows that the GM has three degrees of freedom (3 dof), noting that a gear pair has 2 dof. Alternatively, Talpasanu [START_REF] Talpasanu | Kinematics and Dynamics of Mechanical Systems Based on Graph-Matroid Theory[END_REF] and Tsai [START_REF] Tsai | The kinematics of spatial robotic bevel-gear trains[END_REF] observe that there is a relation between the degrees of freedom f of the GM, the number of links and the number of gear pairs:

f = n -g (1)
again yielding f = 3.

Constraint Analysis via the Matroid Method

To perform the kinematic analysis, we apply the matroid method of Talpasanu [START_REF] Talpasanu | Application of matroid method in kinematic analysis of parallel axes epicyclic gear trains[END_REF] to obtain the Willis kinematic equations for all gear pairs and solve these equations to express all joint variables in terms of the input (sun) variables. This enables us to express the kinematic mapping as a product of exponentials (PoE) in terms of input variables alone and consequently to undertake the singularity analysis. It is worth noting that the Willis equations usually express the relation between angular velocities in a gear-pair/carrier cycle but since the relations between the joint variables

L 0 L 3 L 2 L 4 L 6 L 7 L 1 L 5 L 8 L 9 T 1 T 2 T 3 T 5 T 4 T 6 T 7 T 8 T 9 G 1 G 2 G 3 G 4 G 5 G 6 Fig. 3 Associated digraph
themselves is linear, the same equations hold between the underlying variables as between their velocities. There are essentially three stages to the matroid or incidence-transfer method: the first stage codifies the topology of the digraph representation of the GM in matrix form. The second stage builds the specific design on to this by introducing dimensions that can then be interpreted as gear ratios. The method insures that we obtain a minimal set of linear (Willis) equations and the third stage is to solve these for the joint variables in terms of the input variables.

Associated to the digraph are two matrices. The incidence matrix Π Π Π 0 has rows labelled by the vertices and columns by edges and its entries π 0 i j are -1 or 1 according as edge j leaves or enters vertex i, or else is 0. In this setting, the base L 0 is fixed and its row (containing only -1 and 0) is linearly dependent on the other rows. So, for the purpose of analysis we omit this row and arrive at the reduced incidence matrix Π Π Π as follows:

Π Π Π =              T 1 T 2 T 3 T 4 T 5 T 6 T 7 T 8 T 9 | G 1 G 2 G 3 G 4 G 5 G 6 L 1 1 0 0 -1 -1 -1 -1 0 0 | 0 0 0 0 0 0 L 2 0 1 0 0 0 0 0 0 0 | -1 0 0 0 0 0 L 3 0 0 1 0 0 0 0 0 0 | 0 -1 0 0 0 0 L 4 0 0 0 1 0 0 0 0 0 | 1 0 -1 0 0 0 L 5 0 0 0 0 1 0 0 0 0 | 0 1 0 -1 0 0 L 6 0 0 0 0 0 1 0 0 0 | 0 0 1 0 -1 0 L 7 0 0 0 0 0 0 1 -1 -1 | 0 0 0 1 0 0 L 8 0 0 0 0 0 0 0 1 0 | 0 0 0 0 1 -1 L 9 0 0 0 0 0 0 0 0 1 | 0 0 0 0 0 1              (2)
Further, this is partitioned as indicated into submatrices:

Π Π Π n×m = [P n×t | Pn×g ].
A cycle basis matrix Γ Γ Γ for a digraph consists of a maximally independent set of rows G i , i = 1, . . . , g, each corresponding to a cycle, whose entries γ i j , j = 1, . . . , m are -1 or 1 according as edge j appears in that cycle directed with, or opposed to, a given vertex order for the cycle, or otherwise 0. The cycle space is in fact the nullspace of the incidence matrix so, according to Euler's formula, its dimension is mn. Given the special structure of the digraph for a GM, we have mn = g and a basis for the cycle space can be indexed by the gear pairs, G 1 , . . . , G g . For the given GM, with the vertex order as indicated in Figure 3 by arrows in each basis cycle, we have:

Γ Γ Γ =        T 1 T 2 T 3 T 4 T 5 T 6 T 7 T 8 T 9 | G 1 G 2 G 3 G 4 G 5 G 6 G 1 -1 1 0 -1 0 0 0 0 0 | 1 0 0 0 0 0 G 2 -1 0 1 0 -1 0 0 0 0 | 0 1 0 0 0 0 G 3 0 0 0 1 0 -1 0 0 0 | 0 0 1 0 0 0 G 4 0 0 0 0 1 0 -1 0 0 | 0 0 0 1 0 0 G 5 0 0 0 0 0 1 -1 -1 0 | 0 0 0 0 1 0 G 6 0 0 0 0 0 0 0 1 -1 | 0 0 0 0 0 1        (3)
Again, this can be partitioned into submatrices: Γ Γ Γ g×m = [C g×t |I g×g ] where the second block is the identity matrix. Note that Γ Γ Γ is the cycle basis matrix corresponding to the specific spanning tree for the digraph, which one can obtain by deleting the dashed lines in Figure 3. In any graph with edge set E, the collection I of subsets of E that do not include a cycle form a matroid, mathematical objects that capture the abstract idea of independence. Spanning trees are maximally independent while simple cycles are minimally dependent objects.

The second step is to introduce design parameters into the matrices. The constraint imposed by the cycles on the motion of the GM is captured by the joint position matrix ∆ ∆ ∆ whose entries are δ i j = c i j d i j , where c i j are components of the (reduced) cycle basis matrix C and d i j = y T jy G i where y T j , j = 1, . . . ,t and y G i , i = 1, . . . , g are distances of the axes of turning joint T j and meshing joint G i from the base in home configuration. These distances are the radii of the various gear wheels r k , k = 2, . . . ,t so that:

∆ ∆ ∆ =        T 1 T 2 T 3 T 4 T 5 T 6 T 7 T 8 T 9
G 1 r 2 -r 2 0 -r 4 0 0 0 0 0 G 2 r 3 0 -r 3 0 -r 5 0 0 0 0 G 3 0 0 0 -r 4 0 -r 6 0 0 0 G 4 0 0 0 0 -r 5 0 -r 7 0 0 G 5 0 0 0 0 0 -r 6 r 6 -r 8 0

G 6 0 0 0 0 0 0 0 -r 8 -r 9        (4)
For an oriented gear pair G i , i = 1, . . . , g connecting link (gear wheel) L p to L q , denote the corresponding gear ratio ρ i = -r p /r q . The rows of the matrix represent equations that hold between the joint variables at each revolute pair (or equivalently their angular velocities) so that each row can be independently scaled by one of the radii to realise the gear ratio matrix:

Λ Λ Λ =        T 1 T 2 T 3 T 4 T 5 T 6 T 7 T 8 T 9 G 1 ρ 1 -ρ 1 0 -1 0 0 0 0 0 G 2 ρ 2 0 -ρ 2 0 -1 0 0 0 0 G 3 0 0 0 -ρ 3 0 -1 0 0 0 G 4 0 0 0 0 -ρ 4 0 -1 0 0 G 5 0 0 0 0 0 -ρ 5 ρ 5 -1 0 G 6 0 0 0 0 0 0 0 -ρ 6 -1        (5)
To arrive finally at a complete set of Willis equations for the GM, it is necessary to incorporate the component P of the reduced incidence matrix that provides the connection between the angles of rotation θ i for each link L i and the joint variables φ j at each revolute pair T j , i, j = 1, . . . , n (= t as noted in Section 2). Specifically, set:

Σ Σ Σ g×t = Λ Λ Λ g×t .P T t×t , (6)
then the Willis equations have the matrix form:

Σ Σ Σ.θ θ θ = 0 0 0 (7)
where θ θ θ is the vector of link rotations. We can partition θ θ θ between input variables and passive variables. Following Eq. ( 1), there are three input variables and six passive variables. Explicitly:

θ θ θ = θ θ θ f | θ θ θ g T = θ 1 θ 2 θ 3 | θ 4 θ 5 θ 6 θ 7 θ 8 θ 9 T (8)
Partitioning Σ Σ Σ in a similar way, and expanding the product gives:

Σ Σ Σ = Z Z Z f | Z Z Z g =        L 1 L 2 L 3 L 4 L 5 L 6 L 7 L 8 L 9 G 1 ρ 1 + 1 -ρ 1 0 | -1 0 0 0 0 0 G 2 ρ 2 + 1 0 -ρ 2 | 0 -1 0 0 0 0 G 3 ρ 3 + 1 0 0 | -ρ 3 0 -1 0 0 0 G 4 ρ 4 + 1 0 0 | 0 -ρ 4 0 -1 0 0 G 5 0 0 0 | 0 0 -ρ 5 ρ 5 + 1 -1 0 G 6 0 0 0 | 0 0 0 ρ 6 + 1 -ρ 6 -1        (9)
Now, we can rewrite Eq. ( 7) in the form of

[Z Z Z f | Z Z Z g ].[θ θ θ f | θ θ θ g ] T =
0 0 0. from which it follows that, provided Z Z Z g is non-singular which is easily verified in this case:

[θ θ θ g ] = -[Z Z Z g ] -1 .[Z Z Z f ].[θ θ θ f ] (10)
Solving Eq. ( 10), gives passive variables in terms of input variables as follows:

       θ 4 θ 5 θ 6 θ 7 θ 8 θ 9        =        ρ 1 + 1 -ρ 1 0 ρ 2 + 1 0 -ρ 2 1 -ρ 1 ρ 3 ρ 1 ρ 3 0 1 -ρ 2 ρ 4 0 ρ 2 ρ 4 ρ 1 ρ 3 ρ 5 -ρ 2 ρ 4 (1 + ρ 5 ) + 1 -ρ 1 ρ 3 ρ 5 ρ 2 ρ 4 (1 + ρ 5 ) -ρ 1 ρ 3 ρ 5 ρ 6 + ρ 2 ρ 4 (ρ 5 ρ 6 -1) + 1 ρ 1 ρ 3 ρ 5 ρ 6 -ρ 2 ρ 4 (ρ 5 ρ 6 -1)        .   θ 1 θ 2 θ 3   (11)
4 Kinematic and Singularity Analysis

The forward kinematic map of the mechanism can be written in terms of the revolute pair rotations as a PoE in the relevant Euclidean group (see Murray et al [START_REF] Murray | A Mathematical Introduction to Robotic Manipulation[END_REF]). In this case as the mechanism is planar, the group is SE(2). The form of PoE derives from the corresponding open-loop chain (see Tsai [START_REF] Tsai | Robot Analysis: the Mechanics of serial and parallel manipulators[END_REF]) as follows:

T T T (φ φ φ ) = e X X X 1 φ 1 e X X X 7 φ 7 e X X X 9 φ 9 T T T (0 0 0) (12)
where T T T (0 0 0) is the transformation between base and end-effector frames at the rest position φ φ φ = 0 0 0 and X X X i denote the infinitesimal rotations of revolute joints T i , i = 1, 7, 9 about their centres of rotation. Explicitly, we can use homogeneous representations as follows:

X X X i =   0 -1 0 1 0 -ξ i 0 0 0   (13)
where (with respect to appropriate choices of body coordinates) ξ 1 = 0, ξ 7 = l 1 , ξ 9 = l 1 + l 7 with l 1 = r 2 + 2r 4 + r 6 = r 3 + 2r 5 + r 7 and l 7 = r 6 + 2r 8 + r 9 the lengths of the carrier arms L 1 and L 7 (see Figure 2); and:

T T T (0 0 0) =   1 0 l 1 + l 7 0 1 0 0 0 1   (14)
Then the homogeneous form of the forward kinematic map is

T T T (φ φ φ ) =   cos(φ 1 + φ 7 + φ 9 ) -sin(φ 1 + φ 7 + φ 9 ) l 1 cos φ 1 + l 7 cos(φ 1 + φ 7 ) sin(φ 1 + φ 7 + φ 9 ) cos(φ 1 + φ 7 + φ 9 ) l 1 sin φ 1 + l 7 sin(φ 1 + φ 7 ) 0 0 1   (15)
This can be more simply expressed in terms of link rotation variables using θ 1 = φ 1 , θ 7 = φ 1 + φ 7 , and θ 9 = φ 1 + φ 7 + φ 9 . Moreover, for purposes of singularity analysis it is preferable to work with a local representation of the kinematic mapping T. Simply using the angle θ 9 to parametrise the rotation matrix that constitutes the top left 2 × 2 block of the homogeneous transformation, the local representation is:

(θ 1 , θ 7 , θ 9 ) →   θ 9 l 1 cos θ 1 + l 7 cos θ 7 l 1 sin θ 1 + l 7 sin θ 7   (16)
In this form, we have simply made use of the passive variables that describe the kinematics of the underlying 3R mechanism. These can now be expressed using Eq. ( 11) in terms of the input variables. Hence, the kinematic mapping for the GM can be expressed as a function F : R 3 → R 3 , where:

F (θ 1 , θ 2 , θ 3 ) =   β 1 θ 1 + β 2 θ 2 + β 3 θ 3 l 1 cos θ 1 + l 7 cos(α 1 θ 1 + α 3 θ 3 ) l 1 sin θ 1 + l 7 sin(α 1 θ 1 + α 3 θ 3 )   (17)
and

α 1 = 1 -ρ 2 ρ 4 , α 3 = ρ 2 ρ 4 , β 1 = -ρ 1 ρ 3 ρ 5 ρ 6 + ρ 2 ρ 4 (ρ 5 ρ 6 -1) + 1, β 2 = ρ 1 ρ 3 ρ 5 ρ 6
, and β 3 = -ρ 2 ρ 4 (ρ 5 ρ 6 -1). It is worth noting that by judicious choice of gear ratios the rotation of the end-effector can be made independent of one or more input variables. For example, setting ρ 1 ρ 3 = ρ 5 ρ 6 = 1 (equivalently r 2 = r 6 = r 9 ) ensures the rotation is independent of θ 1 , θ 3 and is directly equal to θ 2 . This is nicely illustrated by Thang [START_REF] Thang | Planar manipulator 3[END_REF].

Finally, to find singularities we need to investigate the Jacobian of the kinematic mapping F . From Equation (17) we obtain:

J =   β 1 β 2 β 3 -l 1 sin θ 1 -α 1 l 7 sin(α 1 θ 1 + α 3 θ 3 ) 0 -α 3 l 7 sin(α 1 θ 1 + α 3 θ 3 ) l 1 cos θ 1 -α 1 l 7 cos(α 1 θ 1 + α 3 θ 3 ) 0 α 3 l 7 cos(α 1 θ 1 + α 3 θ 3 )   (18)
For a singularity, we require:

det(J ) = α 3 β 2 l 1 l 7 sin(θ 1 -α 1 θ 1 -α 3 θ 3 ) = 0. ( 19
)
The design parameters α 3 , β 2 , l 1 , l 7 are assumed non-zero so the GM is singular if and only if sin(θ 1α 1 θ 1α 3 θ 3 ) = 0 and hence:

θ 1 -θ 3 = nπ ρ 2 ρ 4
, for any integer n.

Thus, the singular configurations of mechanism in Figure 1 are strictly contingent on the difference between input variables θ 1 and θ 3 . It can be concluded that increasing the product of gear ratios ρ 2 ρ 4 connecting gear wheels L 3 and L 7 can cause more singular points in the joint space, while keeping it close to zero will reduce singularities. It must be noticed that having ρ 2 ρ 4 1 may have dynamic consequences. The images of the singularity set in the workspace of course correspond to the expected singular configurations in which carrier arms L 1 and L 7 are collinear.

Conclusion

We have illustrated the constraint analysis of a geared mechanism involving two epicyclic gear trains, using the matroid method of Talpasanu. This, in turn, leads to an explicit determination of its passive variables in terms of input variables and thereby to a representation of the kinematic mapping in those variables. Determination of the the singularity set, not only in terms of specific geometric configurations in the workspace-collinearity of the carrier arms-but also in the input space, is a straightforward consequence. This demonstrates the dependency on gear ratios as design parameters for the GM.

Fig. 1 3 -

 3 Fig. 1 3-DoF Geared Planar Manipulator