The Refined Three-Dimensional Structure of Pectate Lyase E from Erwinia chrysanthemi at 2.2 A Resolution

The crystal structure of pectate lyase E (PelE; EC 4.2.2.2) from the enterobacteria Erwinia chrysanthemi has been refined by molecular dynamics techniques to a resolution of 2.2 A and an R factor (an agreement factor between observed structure factor amplitudes) of 16.1%. The final model consists of all 355 amino acids and 157 water molecules. The root-mean-square deviation from ideality is 0.009 A for bond lengths and 1.721[deg] for bond angles. The structure of PelE bound to a lanthanum ion, which inhibits the enzymatic activity, has also been refined and compared to the metal-free protein. In addition, the structures of pectate lyase C (PelC) in the presence and absence of a lutetium ion have been refined further using an improved algorithm for identifying waters and other solvent molecules. The two putative active site regions of PelE have been compared to those in the refined structure of PelC. The analysis of the atomic details of PelE and PelC in the presence and absence of lanthanide ions provides insight into the enzymatic mechanism of pectate lyases.     

The Refined Three-Dimensional Structure of Pectate Lyase C from Erwinia chrysanthemi at 2.2 Angstrom Resolution (Implications for an Enzymatic Mechanism)

The crystal structure of pectate lyase C (EC 4.2.2.2) from the enterobacterium Erwinia chrysanthemi (PelC) has been refined by molecular dynamics techniques to a resolution of 2.2 A to an R factor of 17.97%. The final model consists of 352 of the total 353 amino acids and 114 solvent molecules. The root-mean-square deviation from ideality is 0.009 A for bond lengths and 1.768[deg] for bond angles. The structure of PelC bound to the lanthanide ion lutetium, used as a calcium analog, has also been refined. Lutetium inhibits the enzymatic activity of the protein, and in the PelC-lutetium structure, the ion binds in the putative calcium-binding site. Five side-chain atoms form ligands to the lutetium ion. An analysis of the atomic-level model of the two protein structures reveals possible implications for the enzymatic mechanism of the enzyme.     

Molecular Cloning and Sequencing of the Extracellular Pectate Lyase II Gene from Erwinia carotovora Er

Erwinia carotovora Er produces three extra-cellular pectate lyases (PL I, II, and III). The gene for peetate lyase II (pelII) of E. carotovora Er was cloned and expressed both in Escherichia coli and E. carotovora Er. Localization experiments in E. coli showed that PL II was exclusively in the cytoplasmic space, while PL II was excreted into the culture medium. The complete nucleotides of the pelII gene were sequenced and found to include one open reading frame of 1122 bp coding for a protein of 374 amino acid residues. From comparison of the N-terminal amino acid sequence between the purified PL II and the deduced protein from the nucleotide sequence we reached the conclusion that the mature protein is composed of 352 amino acids with a calculated molecular weight of 38,169 and is preceded by a typical signal sequence of 22 amino acid residues. PL II had 90.1 % and 82.9% homologies with PL I and PL III in amino acid sequence, respectively.     

Multiplication and Virulence in Plant Tissues of Escherichia coli Clones Producing Pectate Lyase Isozymes PLb and PLe at High Levels and of an Erwinia chrysanthemi Mutant Deficient in PLe

The phytopathogenic enterobacterium Erwinia chrysanthemi strain EC16 produces four isozymes of pectate lyase (PL), an extracellular enzyme that macerates parenchymatous plant tissues and kills plant cells. A 1.8-kilobase EcoRI DNA fragment containing the entire pelE gene was deleted from the E. chrysanthemi chromosome by marker exchange of a cloned fragment that had been modified in vitro. The resulting mutant, UM1001, produced the isozymes PLa, PLb, and PLc, but not PLe. Mutant UM1001 was compared with wild-type E. chrysanthemi, with Escherichia coli JA221, and with JA221 containing expression vectors with cloned pel genes producing high levels of PLe (pPEL748) or PLb (pPEL343) for the ability to multiply and cause symptoms in intact potato tubers. Tubers were injected with less than 100 bacteria per inoculation site and incubated aerobically or anaerobically. While maceration occurred only in anaerobically incubated tubers, all of the bacteria, including nonpectolytic E. coli controls, multiplied substantially under all conditions. E. coli JA221(pPEL748) caused significantly more maceration than E. coli JA221(pPEL343) or wild-type E. chrysanthemi. Mutant UM1001 caused significantly less maceration than the wild-type E. chrysanthemi. The results establish the importance of PLe in the pectolytic arsenal of E. chrysanthemi by demonstrating that production of PLe can enable E. coli to aggressively macerate tuber tissue and that deletion of pelE significantly diminishes the virulence of E. chrysanthemi.     

Erwinia amylovora Secretes DspE, a Pathogenicity Factor and Functional AvrE Homolog, through the Hrp (Type III Secretion) Pathway

Erwinia amylovora was shown to secrete DspE, a pathogenicity factor of 198 kDa and a functional homolog of AvrE of Pseudomonas syringae pv. tomato. DspE was identified among the supernatant proteins isolated from cultures grown in an hrp gene-inducing minimal medium by immunodetection with a DspE-specific antiserum. Secretion required an intact Hrp pathway.     

Flavobacterium johnsoniae PorV Is Required for Secretion of a Subset of Proteins Targeted to the Type IX Secretion System

Flavobacterium johnsoniae exhibits gliding motility and digests many polysaccharides, including chitin. A novel protein secretion system, the type IX secretion system (T9SS), is required for gliding and chitin utilization. The T9SS secretes the cell surface motility adhesins SprB and RemA and the chitinase ChiA. Proteins involved in secretion by the T9SS include GldK, GldL, GldM, GldN, SprA, SprE, and SprT. Porphyromonas gingivalis has orthologs for each of these that are required for secretion of gingipain protease virulence factors by its T9SS. P. gingivalis porU and porV have also been linked to T9SS-mediated secretion, and F. johnsoniae has orthologs of these. Mutations in F. johnsoniae porU and porV were constructed to determine if they function in secretion. Cells of a porV deletion mutant were deficient in chitin utilization and failed to secrete ChiA. They were also deficient in secretion of the motility adhesin RemA but retained the ability to secrete SprB. SprB is involved in gliding motility and is needed for formation of spreading colonies on agar, and the porV mutant exhibited gliding motility and formed spreading colonies. However, the porV mutant was partially deficient in attachment to glass, apparently because of the absence of RemA and other adhesins on the cell surface. The porV mutant also appeared to be deficient in secretion of numerous other proteins that have carboxy-terminal domains associated with targeting to the T9SS. PorU was not required for secretion of ChiA, RemA, or SprB, indicating that it does not play an essential role in the F. johnsoniae T9SS.     

Tryptophan-mediated Dimerization of the TssL Transmembrane Anchor Is Required for Type VI Secretion System Activity

The type VI secretion system (T6SS) is a multiprotein complex used by bacteria to deliver effectors into target cells. The T6SS comprises a bacteriophage-like contractile tail structure anchored to the cell envelope by a membrane complex constituted of the TssJ outer-membrane lipoprotein and the TssL and TssM inner-membrane proteins. TssJ establishes contact with the periplasmic domain of TssM whereas the transmembrane segments of TssM and its cytoplasmic domain interact with TssL. TssL protrudes in the cytoplasm but is anchored by a C-terminal transmembrane helix (TMH). Here, we show that TssL TMH dimerization is required for the stability of the protein and for T6SS function. Using the TOXCAT assay and point mutations of the 23 residues of the TssL TMH, we identified Thr194 and Trp199 as necessary for TssL TMH dimerization. NMR hydrogen–deuterium exchange experiments demonstrated the existence of a dimer with the presence of Trp185 and Trp199 at the interface. A structural model based on molecular dynamic simulations shows that TssL TMH dimer formation involves π–π interactions resulting from the packing of the two Trp199 rings at the C-terminus and of the six aromatic rings of Tyr184, Trp185 and Trp188 at the N-terminus of the TMH.     

Basic Amino Acids at the C-Terminus of the Third Intracellular Loop Are Required for the Activation of Phospholipase C by Cholecystokinin-B Receptors

Abstract: In common with other G_q protein-coupled receptors, the third intracellular loop of the cholecystokinin-B (CCK-B) receptor contains three basic amino acids (K333/K334/R335) at the C-terminal segment. To determine the importance of these conserved basic residues in G_q-protein activation and stimulation of phospholipase C, these basic amino acids were mutated. Subsequently, the ability of resulting mutant receptors to activate phospholipase C was investigated by measuring inositol phosphate formation in COS-7 cells and recording Ca²⁺-activated Cl^? currents from Xenopus oocytes. Site-directed mutagenesis was performed to mutate the three basic amino acids, K333/K334/R335, to neutral amino acids, M333/T334/L335. When the resulting mutant CCK-B receptors were expressed in COS-7 cells and Xenopus oocytes, sulfated cholecystokinin octapeptide (CCK-8) failed to induce inositol phosphate formation in COS-7 cells and evoke Ca²⁺-activated Cl^? currents from oocytes. Each basic amino acid was also mutated (K333M, K334T, and R335L). All three single-point mutations resulted in a significant reduction in CCK-8-induced inositol phosphate formation and CCK-8-activated Ca²⁺-dependent Cl^? currents. It is interesting that substituting the basic amino acids, K333/K334/R335, with three other basic residues, R333/R334/K335, did not change the maximal CCK-8-simulated inositol phosphate formation and the amplitude of CCK-8-evoked Ca²⁺-dependent Cl^? currents. Radioligand-binding studies showed that the above-mentioned mutations did not affect the affinity for CCK-8 and receptor expression level in COS-7 cells. These findings suggest that basic amino acids at the C-terminus of the third cytoplasmic loop are required for the signal transduction by CCK-B receptors.     

Type II protein secretion in gram-negative pathogenic bacteria: the study of the structure/secretion relationships of the cellulase Cel5 (formerly EGZ) from Erwinia chrysanthemi

Erwinia chrysanthemi, a Gram-negative plant pathogen, secretes the cellulase Cel5 (formerly EGZ) via the type II secretion pathway (referred to as Out). Cel5 is composed of two domains, a large N-terminal catalytic domain (390 amino acid residues) and a small C-terminal cellulose-binding domain (62 amino acid residues) separated by a linker region. A combination of mutagenesis and structural analysis permitted us to investigate the structure/secretion relationships with respect to the catalytic domain of Cel5. The 3D structure of the catalytic domain was solved by molecular replacement at 2.3 A resolution. Cel5 exhibits the (beta/alpha)8 structural fold and two extra-barrel features. Our previous genetic study based upon tRNA-mediated suppression allowed us to predict positions of importance in the molecule in relation to structure and catalysis. Remarkably, all of the predictions proved to be correct when compared with the present structural information. Mutations of Arg57, which is located at the heart of the catalytic domain, allowed us to test the consequences of structural modifications on the secretion efficiency. The results revealed that secretability imposes remarkably strong constraints upon folding. In particular, an Arg-to-His mutation yielded a species that folded to a stable conformation close to, but distinct from the wild-type, which however was not secretable. We discuss the relationships between folding of a protein in the periplasm, en route to the cell exterior, and presentation of secretion information. We propose that different solutions have been selected for type II secreted exoproteins in order to meet the constraints imposed by their interaction with their respective secretion machineries. We propose that evolutionary pressure has led to the adaptation of different secretion motifs for different type II exoproteins.     

Multiple Heparan Sulfate Binding Site Engagements Are Required for the Infectious Entry of Human Papillomavirus Type 16

Human papillomavirus (HPV) entry is accompanied by multiple receptor-induced conformational changes (CCs) affecting both the major and minor capsid proteins, L1 and L2. Interaction of heparan sulfate (HS) with L1 is essential for successful HPV16 entry. Recently, cocrystallization of HPV16 with heparin revealed four distinct binding sites. Here we characterize mutant HPV16 to delineate the role of engagement with HS binding sites during infectious internalization. Site 1 (Lys278, Lys361), which mediates primary binding, is sufficient to trigger an L2 CC, exposing the amino terminus. Site 2 (Lys54, Lys356) and site 3 (Asn57, Lys59, Lys442, Lys443) are engaged following primary attachment and are required for infectious entry. Site 2 mutant particles are efficiently internalized but fail to undergo an L1 CC on the cell surface and subsequent uncoating in the endocytic compartment. After initial attachment to the cell, site 3 mutants undergo L1 and L2 CCs and then accumulate on the extracellular matrix (ECM). We conclude that the induction of CCs following site 1 and site 2 interactions results in reduced affinity for the primary HS binding site(s) on the cell surface, which allows engagement with site 3. Taken together, our findings suggest that HS binding site engagement induces CCs that prepare the virus for downstream events, such as the exposure of secondary binding sites, CCs, transfer to the uptake receptor, and uncoating.     

The Canonical Twin-Arginine Translocase Components Are Not Required for Secretion of Folded Green Fluorescent Protein from the Ancestral Strain of Bacillus subtilis

Cellular processes, such as the digestion of macromolecules, phosphate acquisition, and cell motility, require bacterial secretion systems. In Bacillus subtilis, the predominant protein export pathways are Sec (generalized secretory pathway) and Tat (twin-arginine translocase). Unlike Sec, which secretes unfolded proteins, the Tat machinery secretes fully folded proteins across the plasma membrane and into the medium. Proteins are directed for Tat-dependent export by N-terminal signal peptides that contain a conserved twin-arginine motif. Thus, utilizing the Tat secretion system by fusing a Tat signal peptide is an attractive strategy for the production and export of heterologous proteins. As a proof of concept, we expressed green fluorescent protein (GFP) fused to the PhoD Tat signal peptide in the laboratory and ancestral strains of B. subtilis. Secretion of the Tat-GFP construct, as well as secretion of proteins in general, was substantially increased in the ancestral strain. Furthermore, our results show that secreted, fluorescent GFP could be purified directly from the extracellular medium. Nonetheless, export was not dependent on the known Tat secretion components or the signal peptide twin-arginine motif. We propose that the ancestral strain contains additional Tat components and/or secretion regulators that were abrogated following domestication.     

TraK and TraB Are Conserved Outer Membrane Proteins of the Neisseria gonorrhoeae Type IV Secretion System and Are Expressed at Low Levels in Wild-Type Cells

Neisseria gonorrhoeae uses a type IV secretion system (T4SS) to secrete chromosomal DNA into the medium, and this DNA is effective in transforming other gonococci via natural transformation. In addition, the T4SS is important in the initial stages of biofilm development and mediates intracellular iron uptake in the absence of TonB. To better understand the mechanism of type IV secretion in N. gonorrhoeae, we examined the expression levels and localization of two predicted T4SS outer membrane proteins, TraK and TraB, in the wild-type strain as well as in overexpression strains and in a strain lacking all of the T4SS proteins. Despite very low sequence similarity to known homologues, TraB (VirB10 homolog) and TraK (VirB9 homolog) localized similarly to related proteins in other systems. Additionally, we found that TraV (a VirB7 homolog) interacts with TraK, as in other T4SSs. However, unlike in other systems, neither TraK nor TraB required the presence of other T4SS components for proper localization. Unlike other gonococcal T4SS proteins we have investigated, protein levels of the outer membrane proteins TraK and TraB were extremely low in wild-type cells and were undetectable by Western blotting unless overexpressed or tagged with a FLAG3 triple-epitope tag. Localization of TraK-FLAG3 in otherwise wild-type cells using immunogold electron microscopy of thin sections revealed a single gold particle on some cells. These results suggest that the gonococcal T4SS may be present in single copy per cell and that small amounts of T4SS proteins TraK and TraB are sufficient for DNA secretion.     

Importance of Type II Secretion for Survival of Legionella pneumophila in Tap Water and in Amoebae at Low Temperatures

Legionella pneumophila type II secretion mutants showed reduced survival in both tap water at 4 to 17°C and aquatic amoebae at 22 to 25°C. Wild-type supernatants stimulated the growth of these mutants, indicating that secreted factors promote low-temperature survival. There was a correlation between low-temperature survival and secretion function when 12 additional Legionella species were examined.     

Requirement of the Type II Secretion System for Utilization of Cellulosic Substrates by Cellvibrio japonicus

Cellulosic biofuels represent a powerful alternative to petroleum but are currently limited by the inefficiencies of the conversion process. While Gram-positive and fungal organisms have been widely explored as sources of cellulases and hemicellulases for biomass degradation, Gram-negative organisms have received less experimental attention. We investigated the ability of Cellvibrio japonicus, a recently sequenced Gram-negative cellulolytic bacterium, to degrade bioenergy-related feedstocks. Using a newly developed biomass medium, we showed that C. japonicus is able to utilize corn stover and switchgrass as sole sources of carbon and energy for growth. We also developed tools for directed gene disruptions in C. japonicus and used this system to construct a mutant in the gspD gene, which is predicted to encode a component of the type II secretion system. The gspD::pJGG1 mutant displayed a greater-than-2-fold decrease in endoglucanase secretion compared to wild- type C. japonicus. In addition, the mutant strain showed a pronounced growth defect in medium with biomass as a carbon source, yielding 100-fold fewer viable cells than the wild type. To test the potential of C. japonicus to undergo metabolic engineering, we constructed a strain able to produce small amounts of ethanol from biomass. Collectively, these data suggest that C. japonicus is a useful platform for biomass conversion and biofuel production.The need for renewable alternatives to petroleum has stimulated interest in many areas of research, including the production of biofuels. Strategies for the production of ethanol from corn starch are well established, but enthusiasm for these approaches is tempered by concerns about cost, as well as indirect effects on global nutrition (38). Cellulosic biofuels represent a potentially useful alternative to starch-based ethanol, in that fuel could be produced from low-value agricultural waste products (10, 39). However, the economic viability of this approach requires overcoming the recalcitrance of plant cell walls to enzymatic degradation (55). This recalcitrance results from the crystalline nature of cellulose, as well as the complexity of plant cell walls, which are a composite of cellulose, hemicellulose, and lignin. As a result, efficient degradation of plant cell walls requires the addition of large quantities of a diverse collection of enzymes, which greatly increases the cost of biomass processing (23).A recent analysis suggested that a combination of consolidated bioprocessing and pretreatment could significantly reduce production costs associated with cellulosic biofuels (48, 52). Consolidated bioprocessing involves the use of a single organism for the degradation of biomass to its component sugars and the subsequent conversion of these sugars to biofuel. Current approaches for the production of consolidated bioprocessors (CBPs) involve the introduction of the genes necessary for ethanol production into organisms capable of deconstructing biomass or the introduction of genes encoding biomass-degrading enzymes and their cognate secretion systems into ethanologenic microorganisms.While Saccharomyces cerevisiae is arguably the most prominent industrial ethanologen, Gram-negative bacteria, such as Zymomonas mobilis, are also used in the commercial production of ethanol (1, 45). Furthermore, studies by the Ingram group have shown that the Gram-negative bacterium Escherichia coli can be engineered for efficient production of ethanol (32). The strong ethanologenic potential of these Gram-negative organisms necessitates the development of technologies for their conversion to consolidated bioprocessors. However, due to differences in cell surface architecture, unique strategies for enzyme display and secretion are required for the construction of consolidated bioprocessors in Gram-negative microorganisms. One approach to overcoming the challenges of secretion/display is to identify Gram-negative organisms that can efficiently degrade bioenergy-relevant biomass substrates. Due to the broad conservation of secretion strategies in Gram-negative microorganisms, the cellulolytic enzymes and secretion machinery from these bacteria would be expected to be transportable to ethanologenic organisms, such as Z. mobilis and E. coli. Consistent with this notion, previous studies have shown that the introduction of the type II secretion system (TTSS) from Erwinia chrysanthemi endowed E. coli with the ability to secrete heterologously expressed E. chysanthemi pectinases (29).Cellvibrio japonicus, originally referred to as Pseudomonas fluorescens var. cellulosa (culture no. 107, isolated in 1948 from soil in Saitama-ken, Japan), has long been known to be capable of cellulose degradation (51). The organism has been reported to produce an extracellular cellulase activity (22, 59), which is secreted into the culture supernatant and does not associate with the cell surface (28). Furthermore, the extracellular cellulases of C. japonicus are inducible, and their activities are increased in the presence of cellulose (35, 40, 60) and strongly downregulated when cellobiose is present in the medium (58).An extensive literature involving the biochemical characterization of C. japonicus polysaccharide-degrading enzymes has been produced over the past 40 years, and in several cases, the corresponding genes have been cloned. Using C. japonicus genomic libraries expressed in E. coli, Wolff et al. described the isolation of four distinct carboxymethylcellulase genes (56). Subsequently, genes encoding endoglucanases, cellodextrinases, xylanases, mannanases, and an arabinofuranosidase have been cloned and characterized (5-8, 17-19, 21, 24). The crystal structures of many of these enzymes have also been published (43, 44). Thus, C. japonicus contains a diverse collection of cellulose-degrading enzymes. The recent analysis of the C. japonicus genome confirmed the presence of an extensive plant cell wall-degrading machinery, predicting the presence of 123 glycoside hydrolases, as well as 14 predicted carbohydrate lyases (15).In this report, we build upon these previous studies and evaluate the ability of C. japonicus to utilize cellulose sources relevant to bioenergy. We demonstrate that C. japonicus can utilize the biomass substrates corn stover (CS) and switchgrass (SG) as sole sources of carbon and energy and show that the bacterium produces acetate, pyruvate, and lactate when grown on monosaccharides and soluble cellulosic carbon sources. We also developed a method for construction of directed gene disruptions in C. japonicus and demonstrate that efficient cellulase secretion and growth on biomass are prevented by disruption of the type II secretion system. In addition, we show that C. japonicus can be metabolically engineered using broad-host-range plasmids. Collectively, our results demonstrate that C. japonicus contains biomass-degrading enzymes and a secretion system that can be used for the engineering of Gram-negative consolidated bioprocessors.     

Organization of Genes Required for the Oxidation of Methanol to Formaldehyde in Three Type II Methylotrophs

Restriction maps of genes required for the synthesis of active methanol dehydrogenase in Methylobacterium organophilum XX and Methylobacterium sp. strain AM1 have been completed and compared. In these two species of pink-pigmented, type II methylotrophs, 15 genes were identified that were required for the expression of methanol dehydrogenase activity. None of these genes were required for the synthesis of the prosthetic group of methanol dehydrogenase, pyrroloquinoline quinone. The structural gene required for the synthesis of cytochrome c(L), an electron acceptor uniquely required for methanol dehydrogenase, and the genes encoding small basic peptides that copurified with methanol dehydrogenases were closely linked to the methanol dehydrogenase structural genes. A cloned 22-kilobase DNA insert from Methylsporovibrio methanica 81Z, an obligate type II methanotroph, complemented mutants that contained lesions in four genes closely linked to the methanol dehydrogenase structural genes. The methanol dehydrogenase and cytochrome c(L) structural genes were found to be transcribed independently in M. organophilum XX. Only two of the genes required for methanol dehydrogenase synthesis in this bacterium were found to be cotranscribed.