Secondary structure and H, N & C resonance assignments of the periplasmic domain of OutG, major pseudopilin from Dickeya dadantii type II secretion system.

The ability to interact and adapt to the surrounding environment is vital for bacteria that colonise various niches and organisms. One strategy developed by Gram-negative bacteria is to secrete exoprotein substrates via the type II secretion system (T2SS). The T2SS is a proteinaceous complex spanning the bacterial envelope that translocates folded proteins such as toxins and enzymes from the periplasm to the extracellular milieu.

Structure-function analysis of pectate lyase Pel3 reveals essential facets of protein recognition by the bacterial type 2 secretion system.

The type II secretion system (T2SS) transports fully folded proteins of various functions and structures through the outer membrane of Gram-negative bacteria. The molecular mechanisms of substrate recruitment by T2SS remain elusive but a prevailing view is that the secretion determinants could be of a structural nature. The phytopathogenic γ-proteobacteria, Pectobacterium carotovorum and Dickeya dadantii, secrete similar sets of homologous plant cell wall degrading enzymes, mainly pectinases, by similar T2SSs, called Out.

The role of intrinsic disorder and dynamics in the assembly and function of the type II secretion system.

Many Gram-negative commensal and pathogenic bacteria use a type II secretion system (T2SS) to transport proteins out of the cell. These exported proteins or substrates play a major role in toxin delivery, maintaining biofilms, replication in the host and subversion of host immune responses to infection. We review the current structural and functional work on this system and argue that intrinsically disordered regions and protein dynamics are central for assembly, exo-protein recognition, and secretion competence of the T2SS.

Dickeya dadantii pectic enzymes necessary for virulence are also responsible for activation of the Arabidopsis thaliana innate immune system.

Soft-rot diseases of plants attributed to Dickeya dadantii result from lysis of the plant cell wall caused by pectic enzymes released by the bacterial cell by a type II secretion system (T2SS). Arabidopsis thaliana can express several lines of defence against this bacterium. We employed bacterial mutants with defective envelope structures or secreted proteins to examine early plant defence reactions.

Bacterial pectate lyases, structural and functional diversity.

Pectate lyases are enzymes involved in plant cell wall degradation. They cleave pectin using a β-elimination mechanism, specific for acidic polysaccharides. They are mainly produced by plant pathogens and plant-associated organisms, and only rarely by animals.

Substrate recognition by the bacterial type II secretion system: more than a simple interaction.

Type II secretion system (T2SS) is a multiprotein trans-envelope complex that translocates fully folded proteins through the outer membrane of Gram-negative bacteria. Although T2SS is extensively studied in several bacteria pathogenic for humans, animals and plants, the molecular basis for exoprotein recruitment by this secretion machine as well as the underlying targeting motifs remain unknown. To address this question, we used bacterial two-hybrid, surface plasmon resonance, in vivo site-specific photo-cross-linking approaches and functional analyses.

Dynamic interplay between the periplasmic and transmembrane domains of GspL and GspM in the type II secretion system.

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.

Anatomy of secretin binding to the Dickeya dadantii type II secretion system pilotin.

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.

PelN is a new pectate lyase of Dickeya dadantii with unusual characteristics.

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.

Cysteine scanning mutagenesis and disulfide mapping analysis of arrangement of GspC and GspD protomers within the type 2 secretion system.

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.

Structural and functional insights into the pilotin-secretin complex of the type II secretion system.

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.

Solution structure of homology region (HR) domain of type II secretion system.

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.

A 20-residue peptide of the inner membrane protein OutC mediates interaction with two distinct sites of the outer membrane secretin OutD and is essential for the functional type II secretion system in Erwinia chrysanthemi.

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).

The crystal structure of pectate lyase peli from soft rot pathogen Erwinia chrysanthemi in complex with its substrate.

The crystallographic structure of the family 3 polysaccharide lyase (PL-3) PelI from Erwinia chrysanthemi has been solved to 1.45 A resolution. It consists of an N-terminal domain harboring a fibronectin type III fold linked to a catalytic domain displaying a parallel beta-helix topology.

Molecular basis of the activity of the phytopathogen pectin methylesterase.

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.

Arabidopsis thaliana expresses multiple lines of defense to counterattack Erwinia chrysanthemi.

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.

The single transmembrane segment drives self-assembly of OutC and the formation of a functional type II secretion system in Erwinia chrysanthemi.

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.

Crystallization of the pectate lyase PelI from Erwinia chrysanthemi and SAD phasing of a gold derivative.

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.

The crystal structure of pectate lyase Pel9A from Erwinia chrysanthemi.

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.

PaeX, a second pectin acetylesterase of Erwinia chrysanthemi 3937.

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.

PehN, a polygalacturonase homologue with a low hydrolase activity, is coregulated with the other Erwinia chrysanthemi polygalacturonases.

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.

Overproduction of the secretin OutD suppresses the secretion defect of an Erwinia chrysanthemi outB mutant.

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.

Functional characterization of the Erwinia chrysanthemi OutS protein, an element of a type II secretion system.

Secretion of pectate lyases and a cellulase occurs in Erwinia 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.