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Neutrinoless double beta decay is a process that violates lepton number conservation. It is predicted to occur in extensions of the standard model of particle physics. This Letter reports the results from phase I of the Germanium Detector Array (GERDA) experiment at the Gran Sasso Laboratory (Italy) searching for neutrinoless double beta decay of the isotope $^{76}\mathrm{Ge}$. Data considered in the present analysis have been collected between November 2011 and May 2013 with a total exposure of 21.6 kg yr. A blind analysis is performed. The background index is about $1\ifmmode\times\else\texttimes\fi{}{10}^{\ensuremath{-}2}\text{ }\text{ }\mathrm{counts}/(\mathrm{keV}\text{ }\mathrm{kg}\text{ }\mathrm{yr})$ after pulse shape discrimination. No signal is observed and a lower limit is derived for the half-life of neutrinoless double beta decay of $^{76}\mathrm{Ge}$, ${T}_{1/2}^{0\ensuremath{\nu}}g2.1\ifmmode\times\else\texttimes\fi{}{10}^{25}\text{ }\text{ }\mathrm{yr}$ (90% C.L.). The combination with the results from the previous experiments with $^{76}\mathrm{Ge}$ yields ${T}_{1/2}^{0\ensuremath{\nu}}g3.0\ifmmode\times\else\texttimes\fi{}{10}^{25}\text{ }\text{ }\mathrm{yr}$ (90% C.L.).

The plant-pathogenic bacterium Dickeya dadantii produces several pectinolytic enzymes that play a major role in the soft-rot disease. Eight characterized endopectate lyases are secreted in the extracellular medium by the type II secretion system, Out. They cleave internal glycosidic bonds of pectin, leading to plant tissue maceration. The D. dadantii pectate lyases belong to different families, namely, PL1, PL2, PL3, and PL9. Analysis of the D. dadantii 3937 genome revealed a gene encoding a new protein of the PL9 family, which already includes the secreted endopectate lyase PelL and the periplasmic exopectate lyase PelX. We demonstrated that PelN is an additional extracellular protein secreted by the Out system. However, PelN has some unusual characteristics. Although most pectate lyases require a very alkaline pH and Ca 2+ for their activity, the PelN activity is optimal at pH 7.4 and in the presence of Fe 2+ as a cofactor. PelN is only weakly affected by the degree of pectin methyl esterification. The PelN structural model, constructed on the basis of the PelL structure, suggests that the PelL global topology and its catalytic amino acids are conserved in PelN. Notable differences concern the presence of additional loops at the PelN surface, and the replacement of PelL charged residues, involved in substrate binding, by aromatic residues in PelN. The pelN expression is affected by different environmental conditions, such as pH, osmolarity, and temperature. It is controlled by the repressors KdgR and PecS and by the activator GacA, three regulators of D. dadantii pectinase genes. Since a pelN mutant had reduced virulence on chicory leaves, the PelN enzyme plays a role in plant infection, despite its low specific activity and its unusual cofactor requirement.

The secretins are a family of large multimeric channels in the outer membrane of Gram-negative bacteria that are involved in protein export. In Dickeya dadantii and many other pathogenic bacteria, the lipoprotein pilotin targets the secretin subunits to the outer membrane, allowing a functional type II secretion system to be assembled. Here, the crystal structure of the C-terminal peptide of the secretin subunit bound to its cognate pilotin is reported. In solution, this C-terminal region of the secretin is nonstructured. The secretin peptide folds on binding to the pilotin to form just under four turns of α-helix which bind tightly up against the first helix of the pilotin so that the hydrophobic residues of the secretin helix can bind to the hydrophobic surface of the pilotin. The secretin helix binds parallel to the first part of the fourth helix of the pilotin. An N-­capping aspartate encourages helix formation and binding by interacting favourably with the helix dipole of the helical secretin peptide. The structure of the secretin–pilotin complex of the phytopathogenic D. dadantii described here is a paradigm for this interaction in the OutS–PulS family of pilotins, which is essential for the correct assembly of the type II secretion system of several potent human adversaries, including enterohaemorrhagic Escherichia coli and Klebsiella oxytoca.

The type II secretion system (T2SS) is a multiprotein nanomachine that transports folded proteins across the outer membrane of gram-negative bacteria. The molecular mechanisms that govern the secretion process remain poorly understood. The inner membrane components GspC, GspL and GspM possess a single transmembrane segment (TMS) and a large periplasmic region and they are thought to form a platform of unknown function. Here, using two-hybrid and pull-down assays we performed a systematic mapping of the GspC/GspL/GspM interaction regions in the plant pathogen Dickeya dadantii. We found that the TMS of these components interact with each other, implying a complex interaction network within the inner membrane. We also showed that the periplasmic, ferredoxin-like, domains of GspL and GspM drive homo- and heterodimerizations of these proteins. Disulfide bonding analyses revealed that the respective domain interfaces include the equivalent secondary-structure elements, suggesting alternating interactions of the periplasmic domains, L/L and M/M versus L/M. Finally, we found that displacements of the periplasmic GspM domain mediate coordinated shifts or rotations of the cognate TMS. These data suggest a plausible mechanism for signal transmission between the periplasmic and the cytoplasmic portions of the T2SS machine.

The type II secretion system (T2SS) secretes enzymes and toxins across the outer membrane of Gram-negative bacteria. The precise assembly of T2SS, which consists of at least 12 core-components called Gsp, remains unclear. The outer membrane secretin, GspD, forms the channels, through which folded proteins are secreted, and interacts with the inner membrane component, GspC. The periplasmic regions of GspC and GspD consist of several structural domains, HR(GspC) and PDZ(GspC), and N0(GspD) to N3(GspD), respectively, and recent structural and functional studies have proposed several interaction sites between these domains. We used cysteine mutagenesis and disulfide bonding analysis to investigate the organization of GspC and GspD protomers and to map their interaction sites within the secretion machinery of the plant pathogen Dickeya dadantii. At least three distinct GspC-GspD interactions were detected, and they involve two sites in HR(GspC), two in N0(GspD), and one in N2(GspD). None of these interactions occurs through static interfaces because the same sites are also involved in self-interactions with equivalent neighboring domains. Disulfide self-bonding of critical interaction sites halts secretion, indicating the transient nature of these interactions. The secretion substrate diminishes certain interactions and provokes an important rearrangement of the HR(GspC) structure. The T2SS components OutE/L/M affect various interaction sites differently, reinforcing some but diminishing the others, suggesting a possible switching mechanism of these interactions during secretion. Disulfide mapping shows that the organization of GspD and GspC subunits within the T2SS could be compatible with a hexamer of dimers arrangement rather than an organization with 12-fold rotational symmetry.

Gram-negative bacteria secrete virulence factors and assemble fibre structures on their cell surface using specialized secretion systems. Three of these, T2SS, T3SS and T4PS, are characterized by large outer membrane channels formed by proteins called secretins. Usually, a cognate lipoprotein pilot is essential for the assembly of the secretin in the outer membrane. The structures of the pilotins of the T3SS and T4PS have been described. However in the T2SS, the molecular mechanism of this process is poorly understood and its structural basis is unknown. Here we report the crystal structure of the pilotin of the T2SS that comprises an arrangement of four α-helices profoundly different from previously solved pilotins from the T3SS and T4P and known four α-helix bundles. The architecture can be described as the insertion of one α-helical hairpin into a second open α-helical hairpin with bent final helix. NMR, CD and fluorescence spectroscopy show that the pilotin binds tightly to 18 residues close to the C-terminus of the secretin. These residues, unstructured before binding to the pilotin, become helical on binding. Data collected from crystals of the complex suggests how the secretin peptide binds to the pilotin and further experiments confirm the importance of these C-terminal residues in vivo., Author Summary Pathogenic bacteria deliver toxins and virulence proteins into host cells and tissues using specialised secretion systems such as the type II and type III secretion systems. These secretion systems have a pore formed by secretin protein subunits through which the disease causing protein effectors and toxins pass. The secretin must be targeted to and assembled in the outer-membrane and a pilotin protein facilitates this process. In the absence of the pilotin the secretin is degraded or mislocates to the inner-membrane, in either case the secretion system in non-functional and the bacterium cannot cause disease. Here we show how the pilotin and the secretin of the type II secretion system interact, these insights may be useful for the development of antibacterial compounds to interfere with secretin targeting and assembly and defeat pathogenic bacteria such as Vibrio cholerae and enterotoxigenic E. coli which infect man and Dickeya dadantii which threatens food security.

The type II secretion system of Gram-negative bacteria is important for bacterial pathogenesis and survival; it is composed of 12 mostly multimeric core proteins, which build a sophisticated secretion machine spanning both bacterial membranes. OutC is the core component of the inner membrane subcomplex thought to be involved in both recognition of substrate and interaction with the outer membrane secretin OutD. Here, we report the solution structure of the HR domain of OutC and explore its interaction with the secretin. The HR domain adopts a β-sandwich-like fold consisting of two β-sheets each composed of three anti-parallel β-strands. This structure is strikingly similar to the periplasmic region of PilP, an inner membrane lipoprotein from the type IV pilus system highlighting the common evolutionary origin of these two systems and showing that all the core components of the type II secretion system have a structural or sequence ortholog within the type IV pili system. The HR domain is shown to interact with the N0 domain of the secretin. The importance of this interaction is explored in the context of the functional secretion system.

The highly stereoselective α(1→4) coupling between two d -galacturonic acid ester derivatives was accomplished in good yields, for the first time, using a phenylthioglycoside as donor. The method was designed to prepare d -galacturonic acid oligomers with methyl ester groups in definite positions.

Methyl (α- d -galactopyranosyluronic acid)-(1→4)- d -galactopyranuronate and methyl α- d -galactopyranosyluronate-(1→4)- d -galactopyranuronic acid have been synthesized by coupling methyl (benzyl 2,3-di-O-benzyl-β- d -galactopyranosid)uronate (3) or benzyl (benzyl 2,3-di-O-benzyl-β- d -galactopyranosid)uronate (4) with benzyl (phenyl 2,3,4-tri-O-benzyl-1-thio-β- d -galactopyranosid)uronate and methyl (phenyl 2,3,4-tri-O-benzyl-1-thio-β- d -galactopyranosid)uronate, respectively, using N-iodosuccinimide and trifluoromethanesulphonic acid as promoters, followed by removal of the benzyl groups. The 4′-OH unprotected dimers benzyl (methyl 2,3-di-O-benzyl-α- d -galactopyranosyluronate)-(1→4)-(benzyl 2,3-di-O-benzyl-β- d -galactopyranosid)uronate and methyl (benzyl 2,3-di-O-benzyl-α- d -galactopyranosyluronate)-(1→4)-(benzyl 2,3-di-O-benzyl-β- d -galactopyranosid)uronate were prepared from methyl (phenyl 2,3-di-O-benzyl-1-thio-4-O-trimethylsilyl-β- d -galactopyranosid)uronate and benzyl (phenyl 2,3-di-O-benzyl-1-thio-4-O-trimethylsilyl-β- d -galactopyranosid)uronate and acceptors 4 or 3, respectively. These compounds have been designed to serve as precursors for the preparation of higher-membered d -galacturonic acid oligomers methyl esterified in definite positions.

The type II secretion system (T2SS) is widely exploited by proteobacteria to secrete enzymes and toxins involved in bacterial survival and pathogenesis. The outer membrane pore formed by the secretin OutD and the inner membrane protein OutC are two key components of the secretion complex, involved in secretion specificity. Here, we show that the periplasmic regions of OutC and OutD interact directly and map the interaction site of OutC to a 20-residue peptide named OutCsip (secretin interacting peptide, residues 139-158). This peptide interacts in vitro with two distinct sites of the periplasmic region of OutD, one located on the N0 subdomain and another overlapping the N2-N3' subdomains. The two interaction sites of OutD have different modes of binding to OutCsip. A single substitution, V143S, located within OutCsip prevents its interaction with one of the two binding sites of OutD and fully inactivates the T2SS. We show that the N0 subdomain of OutD interacts also with a second binding site within OutC located in the region proximal to the transmembrane segment. We suggest that successive interactions between these distinct regions of OutC and OutD may have functional importance in switching the secretion machine.

A quantitative analysis of pectolytic enzymes (polygalacturonase (PG), pectin methyl esterase (PME) and six isoenzymes of pectate lyase (PL)) produced byErwinia bacteria in the presence of diverse carbon sources was made by preparative electrophoresis. Synthesis of each of these enzymes was regulated independently; different induction and repression ratios (about 10- to 1000-fold) were observed for diverse PL isoenzymes, PG and PME. The possibility of using specially constructed media for the production of pectinase complexes with a specific spectra of pectolytic enzymes has been demonstrated.

We provide a mechanism for the activity of pectin methylesterase (PME), the enzyme that catalyses the essential first step in bacterial invasion of plant tissues. The complexes formed in the crystal using specifically methylated pectins, together with kinetic measurements of directed mutants, provide clear insights at atomic resolution into the specificity and the processive action of the Erwinia chrysanthemi enzyme. Product complexes provide additional snapshots along the reaction coordinate. We previously revealed that PME is a novel aspartic-esterase possessing parallel beta-helix architecture and now show that the two conserved aspartates are the nucleophile and general acid-base in the mechanism, respectively. Other conserved residues at the catalytic centre are shown to be essential for substrate binding or transition state stabilisation. The preferential binding of methylated sugar residues upstream of the catalytic site, and demethylated residues downstream, drives the enzyme along the pectin molecule and accounts for the sequential pattern of demethylation produced by both bacterial and plant PMEs.

International audience; Many taxonomically diverse plant species are attacked by Erwinia chrysanthemi, a member of the causal agents of soft-rotting diseases. Symptom development is due to the collective action of pectin-degrading enzymes secreted by the bacterium through a type II secretion system (T2SS). Using Arabidopsis thaliana as a susceptible host, we show that plants respond to E. chrysanthemi 3937 by expressing cell-wall reactions, production of an oxidative burst, and activation of salicylic acid (SA) and jasmonic acid (JA) or ethylene (ET) signaling pathways. We found that the oxidative burst is mainly generated via the expression of the AtrbohD gene, constitutes a barrier of resistance to bacterial attack, and acts independently of the SA-mediated response. To determine the importance of T2SS-secreted proteins in elicitation of these defenses, we used a T2SS deficient mutant and purified enzymatic preparations of representative members of strain 3937 pectate lyase activity. The T2SS-secreted proteins were responsible only partially for the activation of SA and JA or ET signaling pathways observed after infection with the wild-type bacterium and were not involved in the expression of other identified defense reactions. Our study shows the differential role played by pectate lyases isoenzymes in this process and highlights the complexity of the host immune network, which is finely controlled by the bacterium.

Many pathogenic Gram-negative bacteria secrete toxins and lytic enzymes via a multiprotein complex called the type II secretion system. This system, named Out in Erwinia chrysanthemi, consists of 14 proteins integrated or associated with the two bacterial membranes. OutC, a key player in this process, is probably implicated in the recognition of secreted proteins and signal transduction. OutC possesses a short cytoplasmic sequence, a single transmembrane segment (TMS), and a large periplasmic region carrying a putative PDZ domain. A hydrodynamic study revealed that OutC forms stable dimers of an elongated shape, whereas the PDZ domain adopts a globular shape. Bacterial two-hybrid, cross-linking, and pulldown assays revealed that the self-association of OutC is driven by the TMS, whereas the periplasmic region is dispensable for self-association. Site-directed mutagenesis of the TMS revealed that cooperative interactions between three polar residues located at the same helical face provide adequate stability for OutC self-assembly. An interhelical H-bonding mediated by Gln29 appears to be the main driving force, and two Arg residues located at the TMS boundaries are essential for the stabilization of OutC oligomers. Stepwise mutagenesis of these residues gradually diminished OutC functionality and self-association ability. The triple OutC mutant R15V/Q29L/R36A became monomeric and nonfunctional. Self-association and functionality of the triple mutant were partially restored by the introduction of a polar residue at an alternative position in the interhelical interface. Thus, the OutC TMS is more than just a membrane anchor; it drives the protein self-association that is essential for formation of a functional secretion system.

The "family 9 polysaccharide lyase" pectate lyase L (Pel9A) from Erwinia chrysanthemi comprises a 10-coil parallel beta-helix domain with distinct structural features including an asparagine ladder and aromatic stack at novel positions within the superhelical structure. Pel9A has a single high affinity calcium-binding site strikingly similar to the "primary" calcium-binding site described previously for the family Pel1A pectate lyases, and there is strong evidence for a common second calcium ion that binds between enzyme and substrate in the "Michaelis" complex. Although the primary calcium ion binds substrate in subsite -1, it is the second calcium ion, whose binding site is formed by the coming together of enzyme and substrate, that facilitates abstraction of the C5 proton from the sacharride in subsite +1. The role of the second calcium is to withdraw electrons from the C6 carboxylate of the substrate, thereby acidifying the C5 proton facilitating its abstraction and resulting in an E1cb-like anti-beta-elimination mechanism. The active site geometries and mechanism of Pel1A and Pel9A are closely similar, but the catalytic base is a lysine in the Pel9A enzymes as opposed to an arginine in the Pel1A enzymes.

The secretion of extracellular pectinases, among which there are least six isoenzymes of pectate lyase and one pectin methylesterase, allows the phytopathogenic bacterium Erwinia chrysanthemi to degrade pectin. A gene coding for a novel pectin methylesterase has been cloned from an E. chrysanthemi strain 3937 gene library. This gene, pemB, codes for a 433-amino-acid protein. The PemB N-terminal region has the characteristics of lipoprotein signal sequences. We have shown that the PemB precursor is processed and that palmitate is incorporated into the mature protein. The PemB lipoprotein is not released into the extracellular medium and is localized in the outer membrane. The PemB sequence presents homology with other pectin methylesterases from bacterial and plant origin. pemB-like proteins were detected in four other E. chrysanthemi strains but not in Erwinia carotovora strains. PemB was overproduced in Escherichia coli and purified to homogeneity. PemB activity is strongly increased by non-ionic detergents. The enzyme is more active on methylated oligogalacturonides than on pectin, and it is necessary for the growth of the bacteria on oligomeric substrates. PemB is more probably involved in the degradation of methylated oligogalacturonides present in the periplasm of the bacteria, rather than in a direct action on extracellular pectin. pemB expression is inducible in the presence of pectin and is controlled by the negative regulator KdgR.

The pectate lyase PelI is involved in the degradation of plant tissues by the phytopathogenic bacterium Erwinia chrysanthemi. It has been crystallized from a solution containing PEG 550 in the space group P2(1), with unit-cell parameters a = 61.6, b = 70.7, c = 73.4 A, beta = 112.8 degrees. Crystals diffract to 1.45 A using synchrotron radiation. SAD phases have been computed from a gold-derivative crystal at the wavelength of maximum absorption (L(III) edge).

Erwinia chrysanthemi causes soft-rot diseases of various plants by enzymatic degradation of the pectin in plant cell walls. Pectin is a complex polysaccharide. The main chain is constituted of galacturonate residues, and some of them are modified by methyl and/or acetyl esterification. Esterases are necessary to remove these modifications and, thus, to facilitate the further degradation of the polysaccharidic chain. In addition to PaeY, the first pectin acetylesterase identified in the E . chrysanthemi strain 3937, we showed that this bacterium produces a second pectin acetylesterase encoded by the gene paeX . The paeX open reading frame encodes a 322-residue precursor protein of 34,940 Da, including a 21-amino-acid signal peptide. Analysis of paeX transcription, by using gene fusions, revealed that it is induced by pectic catabolic products and affected by catabolite repression. The expression of paeX is regulated by the repressor KdgR, which controls all the steps of pectin catabolism; by the repressor PecS, which controls most of the pectinase genes; and by catabolite regulatory protein, the global activator of sugar catabolism. The paeX gene is situated in a cluster of genes involved in the catabolism and transport of pectic oligomers. In induced conditions, the two contiguous genes kdgM , encoding an oligogalacturonate-specific porin, and paeX are both transcribed as an operon from a promoter proximal to kdgM , but transcription of paeX can also be uncoupled from that of kdgM in noninduced conditions. PaeX is homologous to the C-terminal domain of the Butyrivibrio fibriosolvens xylanase XynB and to a few bacterial esterases. PaeX contains the typical box (GxSxG) corresponding to the active site of the large family of serine hydrolases. Purified PaeX releases acetate from various synthetic substrates and from sugar beet pectin. The PaeX activity increased after previous depolymerization and demethylation of pectin, indicating that its preferred substrates are nonmethylated oligogalacturonides. PaeX is mostly found in the periplasmic space of E . chrysanthemi . These data suggest that PaeX is mainly involved in the deacetylation of esterified oligogalacturonides that enter the periplasm by the KdgM porin.

Erwinia chrysanthemi causes soft-rot diseases of various plants by enzymatic degradation of the pectin in plant cell walls. The structural complexity of pectin requires the combined action of several pectinases for its efficient breakdown. Part of the galacturonate residues of pectin are modified by methylesterification of the carboxyl function and/or acetylesterification of the hydroxyl function in C2 and/or C3 position. Esterases are necessary to cleave these methyl- or acetyl-esterifications. The E. chrysanthemi strain 3937 produces at least two pectin methylesterases (PemA and PemB) and two pectin acetylesterases (PaeY and PaeX). While PaeY is entirely secreted, PaeX is only partially secreted and found both in the extracellular medium and in the periplasm. The two pectin acetylesterases are secreted by the Out system responsible for the secretion of PemA and of eight endo-pectate lyases.

Erwinia chrysanthemi 3937 secretes an arsenal of pectinolytic enzymes, including at least eight endo-pectate lyases encoded by pel genes, which play a major role in the soft-rot disease caused by this bacterium on various plants. E. chrysanthemi also produces some hydrolases that cleave pectin. Three adjacent hydrolase genes, pehV , pehW , and pehX, encoding exo-poly-α- d -galacturonosidases, have been characterized. These enzymes liberate digalacturonides from the nonreducing end of pectin. We report the identification of a novel gene, named pehN, encoding a protein homologous to the glycosyl hydrolases of family 28, which includes mainly polygalacturonases. PehN has a low hydrolase activity on polygalacturonate and on various pectins. PehN action favors the activity of the secreted endo-pectate lyases, mainly PelB and PelC, and that of the periplasmic exo-pectate lyase PelX. However, removal of the pehN gene does not significantly alter the virulence of E. chrysanthemi . Regulation of pehN transcription was analyzed by using gene fusions. Like other pectinase genes, pehN transcription is dependent on several environmental conditions. It is induced by pectic catabolic products and is affected by growth phase, catabolite repression, osmolarity, anaerobiosis, nitrogen starvation, and the presence of calcium ions. The transcription of pehN is modulated by the repressor KdgR, which controls almost all the steps of pectin catabolism, and by cyclic AMP receptor protein (CRP), the global activator of sugar catabolism. The regulator PecS, which represses the transcription of the pel genes but activates that of pehV , pehW , and pehX , also activates transcription of pehN . The three regulators KdgR, PecS, and CRP act by direct interaction with the pehN promoter region. The sequences involved in the binding of these three regulators and of RNA polymerase have been precisely defined. Analysis of the simultaneous binding of these proteins indicates that CRP and RNA polymerase bind cooperatively and that the binding of KdgR could prevent pehN transcription. In contrast, the activator effect of PecS is not linked to competition with KdgR or to cooperation with CRP or RNA polymerase. This effect probably results from competition between PecS and an unidentified repressor involved in peh regulation.

The plant pathogens Erwinia chrysanthemi and Erwinia carotovora secrete multiple exoproteins by a type II pathway, the Out system. Secretion in Erwinia is species-specific: exoproteins of one species cannot be secreted by the other. We analysed the role of two components of the Out system, the bitopic inner membrane protein OutC and the secretin OutD, in the specific recognition of secreted proteins. We demonstrated that the PDZ domain of OutC determines its secretion specificity towards certain exoproteins. The secretin is the major determinant of specificity of the Out system: OutD of E. carotovora changes the secretion specificity of E. chrysanthemi and enables it to secrete heterologous exoproteins. Construction of chimeric OutD showed that the N-terminal region is the specificity domain of the secretin. Thus, both the PDZ domain of OutC and the N-terminal region of OutD are required for specific recognition of secreted proteins. Systematic analysis of the secretion of several exoproteins demonstrated that different exoproteins secreted by the Out machinery have different requirement for their presumed targeting signals on OutC and OutD. This strongly indicates that diverse exoproteins possess a variable number of targeting signals which are recognised by different regions of OutC and OutD.

The plant pathogens Erwinia chrysanthemi and Erwinia carotovora secrete multiple exoproteins by a type II pathway, the Out system. Secretion in Erwinia is species-specific: exoproteins of one species cannot be secreted by the other. We analysed the role of two components of the Out system, the bitopic inner membrane protein OutC and the secretin OutD, in the specific recognition of secreted proteins. We demonstrated that the PDZ domain of OutC determines its secretion specificity towards certain exoproteins. The secretin is the major determinant of specificity of the Out system: OutD of E. carotovora changes the secretion specificity of E. chrysanthemi and enables it to secrete heterologous exoproteins. Construction of chimeric OutD showed that the N-terminal region is the specificity domain of the secretin. Thus, both the PDZ domain of OutC and the N-terminal region of OutD are required for specific recognition of secreted proteins. Systematic analysis of the secretion of several exoproteins demonstrated that different exoproteins secreted by the Out machinery have different requirement for their presumed targeting signals on OutC and OutD. This strongly indicates that diverse exoproteins possess a variable number of targeting signals which are recognised by different regions of OutC and OutD.

OutB is a component of the Erwinia chrysanthemi Out secretion machinery. Homologues of OutB have been described in two other bacteria, Klebsiella oxytoca and Aeromonas hydrophila, but their requirement in the secretion process seems to be different. Study of OutB topology with the BlaM topology probe suggests that it is an inner-membrane protein with a large periplasmic domain. However, fractionation experiments indicate that it could be associated with the outer membrane through its C-terminal part. The secretion deficiency of an Erw. chrysanthemi outB mutant can be reversed by the addition of an inducer of the kdgR regulon. It was shown that this effect results from the increased expression of the secretin OutD and that secretion can be restored in an outB mutant by introducing the outD gene on a plasmid. Several experiments suggest an interaction between OutB and OutD. In Erw. chrysanthemi, the presence of OutD stabilizes OutB. OutD expressed in Escherichia coli can be protected from proteolytic degradation by the coexpression of OutB. This effect does not require the N-terminal, transmembrane segment of outB. OutB can be cross-linked with OutD by formaldehyde. These results indicate that OutB could act with OutD in the functioning of the Out secretion machinery.

Two monomethyl esters of alpha-(1-4)-linked D-galacturonic dimers and three monomethyl esters of alpha-(1-4)-linked D-galacturonic acid trimers were synthesized chemically and further used as substrates in order to establish the substrate specificity of six different endopolygalacturonases from Aspergillus niger, one exopolygalacturonase from Aspergillus tubingensis, and four selected Erwinia chrysanthemi pectinases; exopolygalacturonan hydrolase X (PehX), exopolygalacturonate lyase X (PelX), exopectate lyase W (PelW), and oligogalacturonan lyase (Ogl). All A. niger endopolygalacturonases (PGs) were unable to hydrolyze the two monomethyldigalacturonates and 2-methyltrigalacturonate, whereas 1-methyltrigalacturonate was only cleaved by PGI, PGII, and PGB albeit at an extremely low rate. The hydrolysis of 3-methyltrigalacturonate into 2-methyldigalacturonate and galacturonate by all endopolygalacturonases demonstrates that these enzymes can accommodate a methylgalacturonate at subsite -2. The A. tubingensis exopolygalacturonase hydrolyzed the monomethyl-esterified digalacturonates and trigalacturonates although at lower rates than for the corresponding oligogalacturonates. 1-Methyltrigalacturonate was hydrolyzed at the same rate as trigalacturonate which demonstrates that the presence of a methyl ester at the third galacturonic acid from the nonreducing end does not have any effect on the performance of exopolygalacturonase. Of the four E. chrysanthemi pectinases, Ogl was the only enzyme able to cleave digalacturonate, whereas all four enzymes cleaved trigalacturonate. Ogl does not cleave monomethyl-esterified digalacturonate and trigalacturonate in case the second galacturonic acid residue from the reducing end is methyl-esterified. PehX did not hydrolyze any of the monomethyl-esterified trigalacturonates. The two lyases, PelX and PelW, were both only able to cleave 1-methyltrigalacturonate into Delta4,5-unsaturated 1-methyldigalacturonate and galacturonate.

Five endopectate lyases from the phytopathogenic bacterium Erwinia chrysanthemi , PelA, PelB, PelD, PelI, and PelL, were analyzed with respect to their modes of action on polymeric and oligomeric substrates (degree of polymerization, 2 to 8). On polygalacturonate, PelB showed higher reaction rates than PelD, PelI, and PelA, whereas the reaction rates for PelL were extremely low. The product progression during polygalacturonate cleavage showed a typical depolymerization profile for each enzyme and demonstrated their endolytic character. PelA, PelI, and PelL released oligogalacturonates of different sizes, whereas PelD and PelB released mostly unsaturated dimer and unsaturated trimer, respectively. Upon prolonged incubation, all enzymes degraded the primary products further, to unsaturated dimer and trimer, except for PelL, which degraded the primary products to unsaturated tetramer and pentamer in addition to unsaturated dimer and trimer. The bond cleavage frequencies on oligogalacturonates revealed differences in the modes of action of these enzymes that were commensurate with the product progression profiles. The preferential products formed from the oligogalacturonates were unsaturated dimer for PelD, unsaturated trimer for PelB, and unsaturated tetramer for PelI and PelL. For PelA, preferential products were dependent on the sizes of the oligogalacturonates. Whereas PelB and PelD displayed their highest activities on hexagalacturonate and tetragalacturonate, respectively, PelA, PelI, and PelL were most active on the octamer, the largest substrate used. The bond cleavage frequencies and reaction rates were used to estimate the number of subsites of each enzyme.

Methyl (alpha-D-galactopyranosyluronic acid)-(1--4)-D-galactopyranuronate and methyl alpha-D-galactopyranosyl-uronate-(1--4)-D-galactopyranuronic acid have been synthesized by coupling methyl (benzyl 2,3-di-O-benzyl-beta-D-galactopyranosid)uronate (3) or benzyl (benzyl 2,3-di-O-benzyl-beta-D-galactopyranosid)uronate (4) with benzyl (phenyl 2,3,4-tri-O-benzyl-1-thio-beta-D-galactopyranosid)uronate and methyl (phenyl 2,3,4-tri-O-benzyl-1-thio-beta-D-galactopyranosid)uronate, respectively, using N-iodosuccinimide and trifluoromethanesulphonic acid as promoters, followed by removal of the benzyl groups. The 4'-OH unprotected dimers benzyl (methyl 2,3-di-O-benzyl-alpha-D-galactopyranosyluronate)-(1--4)-(benzyl 2,3-di-O-benzyl-beta-D-galactopyranosid)uronate and methyl (benzyl 2,3-di-O-benzyl-alpha-D-galactopyranosyluronate)-(1--4)-(benzyl 2,3-di-O-benzyl-beta-D-galactopyranosid)uronate were prepared from methyl (phenyl 2,3-di-O-benzyl-1-thio-4-O-trimethylsilyl-beta-D-galactopyranosid) uronate and benzyl (phenyl 2,3-di-O-benzyl-1-thio-4-O-trimethylsilyl-beta-D-galactopyranosid) uronate and acceptors 4 or 3, respectively. These compounds have been designed to serve as precursors for the preparation of higher-membered D-galacturonic acid oligomers methyl esterified in definite positions.

SUMMARY: Secretion of pectate lyases and a cellulase occurs in winis chrysanthemi through a type II secretion machinery, the Out system. Proper insertion of the secretin OutD in the outer membrane requires the presence of Outs. Outs is an outer-membrane lipoprotein that interacts directly with OutD. Using ligand-blotting experiments, it has been shown that this interaction requires at least the 62 C-terminal amino acids of OutD. When this domain was added to the C-terminal extremity of the secreted pectate lyase PelD, the construct was stabilized by Outs but not inserted into the outer membrane. Thus, this domain is sufficient to interact with Outs but it is unable to confer the ability to be inserted into the outer membrane in the presence of Outs. A screen for outs mutants unable to secrete pectate lyases gave only mutants unable to properly localize OutD in the outer membrane and no mutant in the protection function. Thus, the interaction between Outs and OutD can probably not be abolished by the mutation of a single amino acid, and the insertion of OutD in the outer membrane may require additional proteins.

PeIL, a pectate lyase (E.C. 4.2.2.9) from E. chrysanthemi 3937 that is not homologous to the lyases with known structures, has been purified and crystallized by the hanging-drop method using a variety of organic solvents as precipitant. Elongated lathes grown from poly(ethylene glycol) plus isopropanol belong to the space group P212121 with cell dimensions a = 55.5, b = 58.2, c = 16.4 Å with a single molecule in the asymmetric unit. Although complete data sets have been collected to 2.3 Å resolution, these crystals diffract to at least 1.9 Å resolution and are suitable for structure determination. Chunky plates grown using other organic solvents as the precipitant diffracted to 3 Å resolution and were partially characterized as a second orthorhombic crystal form with space group P21212 and cell dimensions a = 119.1, b = 140.5 and c = 105.4 Å, suggesting four molecules in the asymmetric unit.

OutD is an outer membrane component of the main terminal branch of the general secretory pathway (GSP) in Erwinia chrysanthemi. We analyzed the interactions of OutD with other components of the GSP (Out proteins) and with secreted proteins (PelB, EGZ and PemA). OutD is stabilized by its interaction with another GSP component, OutS. The 62 C-terminal amino acids of OutD are necessary for this interaction. In vivo formation of OutD multimers, up to tetramers, was proved after the dissociation in mild conditions of the OutD aggregates formed in the outer membrane. Thus, OutD could form a channel-like structure in the outer membrane. We showed that OutD is stabilized in vivo when co-expressed with Out-secreted proteins. This stabilization results from the formation of complexes that were detected in experiments of co-immunoprecipitation and co-sedimentation in sucrose density gradients. The presence of the N-terminal part of OutD is required for this interaction. The interaction between OutD and the secreted protein PelB was confirmed in vitro, suggesting that no other component of the GSP is required for this recognition. No interaction was observed between the E. carotovora PelC and the E. chrysanthemi OutD. Thus, the interaction between GspD and the secreted proteins present in the periplasm could be the key to the specificity of the secretion machinery and a trigger for that process.

The highly stereoselective α(1→4) coupling between two d -galacturonic acid ester derivatives was accomplished in good yields, for the first time, using a phenylthioglycoside as donor. The method was designed to prepare d -galacturonic acid oligomers with methyl ester groups in definite positions.

The bacteria Erwinia chrysanthemi secretes in the extracellular medium several pectinases of different types. A gene coding for a novel pectin methylesterase pemB has been cloned from an E. chrysanthemi strain 3937 gene library. The PemB sequence presents homology with other pectin methylesterases from bacteria and plants. PemB has been characterised as a bacterial lipoprotein. PemB is localised in the outer membrane and is not released into the extracellular medium. PemB was overproduced in E. coli and purified to homogeneity. It is activated by non-ionic detergents. PemB has a highest activity on methylated oligogalacturonides and this enzyme is necessary for the growth of the bacteria on this substrate as carbon source.

Summary An Erwinia chrysanthemi gene able to complement an Escherichia coli dsbA mutation has been cloned and sequenced. This gene codes for a periplasmic protein with disulphide isomerase activity that has 69% identity and 94% similarity with the E. coli DsbA protein. An E. chrysanthemi dsbA-uidA fusion mutant has been constructed. dsbA expression seems to be constitutive. This mutant has multiple phenotypes resulting from the absence of disulphide bond formation in periplasmic and secreted proteins. Pectate lyases and the cellulase EGZ are rapidly degraded in the periplasm of the dsbA mutant. E. chrysanthemi synthesizes another periplasmic protein with disulphide isomerase activity, namely DsbC. The dsbC gene introduced on a multicopy plasmid in a dsbA mutant was only partially able to restore EGZ secretion, indicating that even if DsbA and DsbC possess disulphide oxydoreductase activity, they are not completely interchangeable. Moreover, pectate lyases expressed in an E. coli dsbA mutant were very instable but their stability was unaffected in a dsbC mutant. These results indicate that DsbA and DsbC could have different substrate specificities.

International audience; We identified and characterized an Erwinia chrysanthemi gene able to complement an Escherichia coli dsbA mutation that prevents disulfide bond formation in periplasmic proteins. This gene, dsbC, codes for a 24 kDa periplasmic protein that contains a characteristic active site sequence of disulfide isomerases, Phe-X-X-X-X-Cys-X-X-Cys. Besides the active site, DsbC has no homology with DsbA, thioredoxin or eukaryotic protein disulfide isomerase and it could define a new subfamily of disulfide isomerases. Purified DsbC protein is able to catalyse insulin oxidation in a dithiothreitol dependent manner. The E.coli gene xprA codes for a protein functionally equivalent to DsbC. The in vivo function of DsbC seems to be the formation of disulfide bonds in proteins. The presence of XprA could explain the residual disulfide isomerase activity existing in dsbA mutants. Re-oxidation of XprA does not seem to occur through DsbB, the protein that probably re-oxidizes DsbA.

The GERDA (GERmanium Detector Array) is an experiment for the search of neutrinoless double beta decay ( 0 ν β β ) in 76Ge, located at Laboratori Nazionali del Gran Sasso of INFN (Italy). In the first phase of the experiment, a 90% confidence level (C.L.) sensitivity of 2.4 ⋅ 10 25 yr on the 0 ν β β decay half-life was achieved with a 21.6 kg⋅yr exposure and an unprecedented background index in the region of interest of 10−2 counts/(keV⋅kg⋅yr). No excess of signal events was found, and an experimental lower limit on the half-life of 2.1 ⋅ 1025 yr (90% C.L.) was established. Correspondingly, the limit on the effective Majorana neutrino mass is m e e 0.2 – 0.4 eV , depending on the considered nuclear matrix element. The previous claim for evidence of a 0 ν β β decay signal is strongly disfavored, and the field of research is open again.

Erwinia chrysanthemi DNA fragment digested by restriction endonuclease EcoRI and carrying the gene EC16 determining the synthesis of pectatelyase with Rf 0.20 and mol. mass 40kD has been cloned in plasmid pUC 9 plasmid in Escherichia coli HB101 cells. Three genes for pectatelyases of Erwinia chrysanthemi ENA49 have been cloned in vector phage lambda 47.1 in Escherichia coli cells. Two genes determining the synthesis of pectatelyases with Rf 0.06 and 0.19 and mol. masses 40 kD and 39 kD have been cloned as a part of an 7 kb Eco RI-fragment, that suggested their close location on the chromosome of Erwinia chrysanthemi ENA49. All of the cloned pectatelyase genes are expressed constitutively with pectatelyases accumulating in periplasm and being unable to secret into the cultural medium.

The GERDA collaboration is performing a search for neutrinoless double beta decay of 76Ge with the eponymous detector. The experiment has been installed and commissioned at the Laboratori Nazionali del Gran Sasso and has started operation in November 2011. The design, construction and first operational results are described, along with detailed information from the R&D phase. peerReviewed

The GERmanium Detector Array (Gerda) experiment at the Gran Sasso underground laboratory (LNGS) of INFN is searching for neutrinoless double beta ( $$0\nu \beta \beta $$ ) decay of $$^{76}$$ Ge. The signature of the signal is a monoenergetic peak at 2039 keV, the $$Q_{\beta \beta }$$ value of the decay. To avoid bias in the signal search, the present analysis does not consider all those events, that fall in a 40 keV wide region centered around $$Q_{\beta \beta }$$ . The main parameters needed for the $$0\nu \beta \beta $$ analysis are described. A background model was developed to describe the observed energy spectrum. The model contains several contributions, that are expected on the basis of material screening or that are established by the observation of characteristic structures in the energy spectrum. The model predicts a flat energy spectrum for the blinding window around $$Q_{\beta \beta }$$ with a background index ranging from 17.6 to 23.8 $$\times $$ $$10^{-3}$$ cts/(keV kg yr). A part of the data not considered before has been used to test if the predictions of the background model are consistent. The observed number of events in this energy region is consistent with the background model. The background at $$Q_{\beta \beta }$$ is dominated by close sources, mainly due to $$^{42}$$ K, $$^{214}$$ Bi, $$^{228}$$ Th, $$^{60}$$ Co and $$\alpha $$ emitting isotopes from the $$^{226}$$ Ra decay chain. The individual fractions depend on the assumed locations of the contaminants. It is shown, that after removal of the known $$\gamma $$ peaks, the energy spectrum can be fitted in an energy range of 200 keV around $$Q_{\beta \beta }$$ with a constant background. This gives a background index consistent with the full model and uncertainties of the same size.

Erwinia atroseptica 36A cells were transformed by the recombinant plasmid pPL5-1 (a derivative of the vector plasmid pUC19) containing pelb and pelc genes which encode pectate lyases of Erwinia chrysanthemi ENA49. Synthesis of pectate lyases PLB and PLC determined by the cloned pel genes is constitutive in Erwinia atroseptica 36ApPL5-1 cells and not inducible by sodium polypectate. The major part of these enzymes was accumulated in the periplasmic fraction of Erwinia atroseptica and cells were unable to efficiently secrete the enzymes into the cultural medium. Synthesis and secretion of the native pectate lyases by Erwinia atroseptica harboring the plasmid were as efficient as by the parental cells. The obtained results suggest the high specificity of pectate lyase secretory systems of kindred Erwinias.

Erwinia chrysanthemi 3937 secretes into the external medium several pectinolytic enzymes, among which are eight isoenzymes of the endo-cleaving pectate lyases: PelA, PelB, PelC, PelD, and PelE (family 1); PelI (family 4); PelL (family 3); and PelZ (family 5). In addition, one exo-cleaving pectate lyase, PelX (family 3), has been found in the periplasm of E. chrysanthemi . The E. chrysanthemi 3937 gene kdgC has been shown to exhibit a high degree of similarity to the genes pelY of Yersinia pseudotuberculosis and pelB of Erwinia carotovora , which encode family 2 pectate lyases. However, no pectinolytic activity has been assigned to the KdgC protein. After verification of the corresponding nucleotide sequence, we cloned a longer DNA fragment and showed that this gene encodes a 553-amino-acid protein exhibiting an exo-cleaving pectate lyase activity. Thus, the kdgC gene was renamed pelW . PelW catalyzes the formation of unsaturated digalacturonates from polygalacturonate or short oligogalacturonates. PelW is located in the bacterial cytoplasm. In this compartment, PelW action could complete the degradation of pectic oligomers that was initiated by the extracellular or periplasmic pectinases and precede the action of the cytoplasmic oligogalacturonate lyase, Ogl. Both cytoplasmic pectinases, PelW and Ogl, seem to act in sequence during oligogalacturonate depolymerization, since oligomers longer than dimers are very poor substrates for Ogl but are good substrates for PelW. The estimated number of binding subsites for PelW is three, extending from subsite −2 to +1, while it is probably two for Ogl, extending from subsite −1 to +1. The activities of the two cytoplasmic lyases, PelW and Ogl, are dependent on the presence of divalent cations, since both enzymes are inhibited by EDTA. In contrast to the extracellular pectate lyases, Ca 2+ is unable to restore the activity of PelW or Ogl, while several other cations, including Co 2+ , Mn 2+ , and Ni 2+ , can activate both cytoplasmic lyases.

The gene for a pectate lyase of E. chrysanthemi ENA49 cloned in a recombinant plasmid pPTL1 (a derivative of RSF1010) was transferred into E. carotovora. The pectate lyase determined by the cloned gene was secreted into the cultural medium from the cells of E. crysanthemi EC16. Partial secretion of the enzyme was registered for E. carotovora cells. The major part of EC1 E. chrysanthemi pectate lyase synthesized by E. carotovora cells is accumulated in periplasmic and cytoplasmic fractions. The obtained results suggest the different specificity or efficiency of pectate lyase secretion systems in the studied Erwinia strains.

The combined changes (specters of isoenzymes of intracellular and extracellular pectatelyase, protein composition of periplasm and outer membrane) in the cells of E. chrysanthemi ENA49 from the periodical culture growing on the inducer containing medium have been studied. The beginning of active pectatelyase synthesis was established to be accompanied by the temporal intracellular accumulation of the enzyme. The cellular capability of pectatelyase secretion to the outer medium correlates with the presence of the definite protein in the outer membrane. The different pathways for secretion of pectatelyase isoenzymes PLb, PLc, PLd and PLe are suggested.

E.atroseptica 36A cells were transformed by the recombinant plasmids p27-1 and pEA364 (derivatives of the vector plasmid pUC19) containing pectate lyase genes of E.carotovora 17A and E.atroseptica 36A, respectively. The synthesis of pectate lyases determined by the cloned genes of bacteria of both subspecies, as well as the synthesis of the native enzymes, were induced by sodium poly pectate. Increase of the dose of pectate lyase genes did not result in alteration of pectate lyase secretion by E.atroseptica 36ApEA364 cells. At the same time, the efficiency of secretion of heterologous pectate lyases by E.atroseptica 36Ap27-1 cells was lower. The synthesis and secretion of the resident isoenzymes are as efficient as those of the parental cells. The results indicate a high specificity of the pectinase secretory system in Erwinia of different species and, moreover, subspecies.