Mutagenesis of cellulase EGZ for studying the general protein secretory pathway in Erwinia chrysanthemi

Extracellular secretion of endoglucanase Z (EGZ) from Erwinia chrysanthemi is mediated by the so-called Out general secretion pathway and, presumably, involves recognition of EGZ-carried structural information by one or more of the Out proteins. Investigating the relationships between structure and secretability of EGZ was the purpose of the present work. EGZ is made of two independent domains, located at the N-and C-proximal sides, separated by a Ser/Thr-rich region, which are responsible for catalysis and cellulose-binding, respectively. The existence of a secretion region (‘targeting signal’) was investigated by studying the secretability of modified EGZ derivatives. These resulted from deletion or peptide insertion and were designed by using the domain organization cited above as a guide. Catalytic and/or cellulose-binding tests showed that all proteins exhibited at least a functional EGZ domain while immunoblot analyses confirmed that neither the insertions nor the deletions led to grossly misfolded proteins. In contrast, all of the proteins lost their secretability in E. chrysanthemi. This suggested that at least two secretion motifs existed, one lying within each functional domain. The role of the Ser/Thr-rich linker region was subsequently tested. Accordingly, two proteins containing a linker region whose length was increased by the addition of 8 and 18 additional residues and one protein lacking the linker region were studied. All three exhibited endoglucanase activity and cellulose-binding ability, confirming the independence of the domains within the context of EGZ/polysaccharide interaction. In contrast, none was secreted by E. chrysanthemi. Collectively, our results with EGZ (i) suggest the occurrence of multiple secretion-related sites either acting sequentially or forming a single three-dimensional secretion signal, (ii) show that secretability is not determined by either one of the two functional domains alone, and (iii) reveal that the linker region plays a role in secretion. We propose that all EGZ derivatives were impaired in the recognition step, the nature of which is discussed.     

Alteration of a single tryptophan residue of the cellulose-binding domain blocks secretion of the Erwinia chrysanthemi Cel5 cellulase (ex-EGZ) via the type II system

Cel5 (formerly known as endoglucanase Z) of Erwinia chrysanthemi is secreted by the Out type II pathway. Previous studies have shown that the catalytic domain (CD), linker region (LR) and cellulose-binding domain (CBD) each contain information needed for secretion. The aim of this work was to further investigate the secretion-related information present in the CBD(Cel5). Firstly(, )deleting a surface-exposed flexible loop had no effect on secretion. This indicated that some structural freedom is tolerated by the type II system. Secondly, mutation of a single tryptophan residue, previously shown to be important for binding to cellulose, i.e. Trp43, was found also to impair secretion. This indicated that the flat cellulose-binding surface of CBD(Cel5 )contains secretion-related information. Thirdly, CBD(Cel5) was substituted by the CBD(EGG) of Alteromonas haloplanctis endoglucanase G, yielding a hybrid protein CD(Cel5)-LR(Cel5)-CBD(EGG) that exhibited 90 % identity with Cel5, including the Trp43 residue. The hybrid protein was not secreted. This indicated that the Trp43 residue is necessary but not sufficient for secretion. Here we propose a model in which the secretion of Cel5 involves a transient intramolecular interaction between the cellulose-binding surface of CBD(Cel5) and a region close to the entry into the active site in CD(Cel5). Once secreted, the protein may then open out to allow the cellulose-binding surface of CBD(Cel5 )to interact with the surface of the cellulose substrate. An implication of this model is that protein molecules fold to a specific secretion-competent conformation prior to secretion that is different from the folding state of the secreted species.     

Construction of minimum size cellulase (Cel5Z) from Pectobacterium chrysanthemi PY35 by removal of the C-terminal region

Pectobacterium chrysanthemi PY35 secretes the endoglucanase Cel5Z, an enzyme of the glycoside hydrolase family 5. Cel5Z is a 426 amino acid, signal peptide (SP)-containing protein composed of two domains: a large N-terminal catalytic domain (CD; 291 amino acids) and a small C-terminal cellulose binding domain (CBD; 62 amino acids). These two domains are separated by a 30 amino acid linker region (LR). A truncated cel5Z gene was constructed with the addition of a nonsense mutation that removes the C-terminal region of the protein. A truncated Cel5Z protein, consisting of 280 amino acid residues, functioned as a mature enzyme despite the absence of the SP, 11 amino acid CD, LR, and CBD region. In fact, this truncated Cel5Z protein showed an enzymatic activity 80% higher than that of full-length Cel5Z. However, cellulase activity was undetectable in mature Cel5Z proteins truncated to less than 280 amino acids.     

Protein secretion in gram-negative bacteria. The extracellular metalloprotease B from Erwinia chrysanthemi contains a C-terminal secretion signal analogous to that of Escherichia coli alpha-hemolysin

The secretion signal of extracellular metalloprotease B that is secreted without a signal peptide by the Gram-negative phytopathogenic bacterium Erwinia chrysanthemi is shown by deletion and gene fusion analyses to be located within the last 40 C-terminal amino acids. Secretion of a peptide containing only this region of the protease requires the same three secretion factors (PrtD, PrtE, and PrtF) that were previously shown to be required for the secretion of the full-length protease. This secretion signal can also be recognized, albeit inefficiently, by the analogous secretion machinery of alpha-hemolysin, another protein with a C-terminal secretion signal that is secreted by some strains of the Gram-negative bacterium Escherichia coli. The secretion signal was fused to an internal 200-amino acid fragment from the sequence of the cytoplasmic protein amylomaltase to promote its specific secretion by the protease secretion pathway. Almost exactly the same sequence as that identified as the protease B secretion signal was also found at the C terminus of metalloprotease C that is also secreted by E. chrysanthemi.     

Crystal structure of the cellulase Cel9M enlightens structure/function relationships of the variable catalytic modules in glycoside hydrolases

Cellulases cleave the beta-1.4 glycosidic bond of cellulose. They have been characterized as endo or exo and processive or nonprocessive cellulases according to their action mode on the substrate. Different types of these cellulases may coexist in the same glycoside hydrolase family, which have been classified according to their sequence homology and catalytic mechanism. The bacterium C. celluloyticum produces a set of different cellulases who belong mostly to glycoside hydrolase families 5 and 9. As an adaptation of the organism to different macroscopic substrates organizations and to maximize its cooperative digestion, it is expected that cellulases of these families are active on the various macroscopic organizations of cellulose chains. The nonprocessive cellulase Cel9M is the shortest variant of family 9 cellulases (subgroup 9(C)) which contains only the catalytic module to interact with the substrate. The crystal structures of free native Cel9M and its complex with cellobiose have been solved to 1.8 and 2.0 A resolution, respectively. Other structurally known family 9 cellulases are the nonprocessive endo-cellulase Cel9D from C. thermocellum and the processive endo-cellulase Cel9A from T. fusca, from subgroups 9(B1) and 9(A), respectively, whose catalytic modules are fused to a second domain. These enzymes differ in their activity on substrates with specific macroscopic appearances. The comparison of the catalytic module of Cel9M with the two other known GH family 9 structures may give clues to explain its substrate profile and action mode.     

Type 1 protein secretion in bacteria, the ABC-transporter dependent pathway (review)

The relatively simple type 1 secretion system in gram-negative bacteria is nevertheless capable of transporting polypeptides of up to 800 kDa across the cell envelope in a few seconds. The translocator is composed of an ABC-transporter, providing energy through ATP hydrolysis (and perhaps the initial channel across the inner membrane), linked to a multimeric Membrane Fusion Protein (MFP) spanning the initial part of the periplasm and forming a continuous channel to the surface with an outer membrane trimeric protein. Proteins targeted to the translocator carry an (uncleaved), poorly conserved secretion signal of approximately 50 residues. In E. coli the HlyA toxin interacts with both the MFP (HlyD) and the ABC protein HlyB, (a half transporter) triggering, via a conformational change in HlyD, recruitment of the third component, TolC, into the transenvelope complex. In vitro, HlyA, through its secretion signal, binds to the nucleotide binding domain (NBD or ABC-ATPase) of HlyB in a reaction reversible by ATP that may mimic initial movement of HlyA into the translocation channel. HlyA is then transported rapidly, apparently in an unfolded form, to the cell surface, where folding and release takes place. Whilst recent structural studies of TolC and MFP-like proteins are providing atomic detail of much of the transport path, structural analysis of the HlyB NBD and other ABC ATPases, have revealed details of the catalytic cycle within an NBD dimer and a glimpse of how the action of HlyB is coupled to the translocation of HlyA.     

Protein secretion in the absence of ATP: the autotransporter, two-partner secretion and chaperone/usher pathways of gram-negative bacteria (review)

Bacteria secrete a wide variety of proteins, many of which play important roles in virulence. In gram-negative bacteria, these proteins must cross the cytoplasmic or inner membrane, periplasm, and outer membrane to reach the cell surface. Gram-negative bacteria have evolved multiple pathways to allow protein secretion across their complex envelope. ATP is not available in the periplasm and many of these secretion pathways encode components that harness energy available at the inner membrane to drive secretion across the outer membrane. In contrast, the autotransporter, two-partner secretion and chaperone/usher pathways are comparatively simple systems that allow secretion across the outer membrane without the need for input of energy from the inner membrane. This review will present overviews of these 'self-sufficient' pathways, focusing on recent advances and secretion mechanisms. Similarities among the pathways and with other protein translocation mechanisms will be highlighted.     

Enhancement of expression and apparent secretion of Erwinia chrysanthemi endoglucanase (encoded by celZ) in Escherichia coli B.

Escherichia coli B has been engineered as a biocatalyst for the conversion of lignocellulose into ethanol. Previous research has demonstrated that derivatives of E. coli B can produce high levels of Erwinia chrysanthemi endoglucanase (encoded by celZ) as a periplasmic product and that this enzyme can function with commercial fungal cellulase to increase ethanol production. In this study, we have demonstrated two methods that improve celZ expression in E. coli B. Initially, with a low-copy-number vector, two E. coli glycolytic gene promoters (gap and eno) were tested and found to be less effective than the original celZ promoter. By screening 18,000 random fragments of Zymomonas mobilis DNA, a surrogate promoter was identified which increased celZ expression up to sixfold. With this promoter, large polar inclusion bodies were clearly evident in the periplasmic space. Sequencing revealed that the most active surrogate promoter is derived from five Sau3A1 fragments, one of which was previously sequenced in Z. mobilis. Visual inspection indicated that this DNA fragment contains at least five putative promoter regions, two of which were confirmed by primer extension analysis. Addition of the out genes from E. chrysanthemi EC16 caused a further increase in the production of active enzyme and facilitated secretion or release of over half of the activity into the extracellular environment. With the most active construct, of a total of 13,000 IU of active enzyme per liter of culture, 7,800 IU was in the supernatant. The total active endoglucanase was estimated to represent 4 to 6% of cellular protein.     

Type II protein secretion in Pseudomonas aeruginosa: the pseudopilus is a multifibrillar and adhesive structure

The type II secretion pathway of Pseudomonas aeruginosa is involved in the extracellular release of various toxins and hydrolytic enzymes such as exotoxin A and elastase. This pathway requires the function of a macromolecular complex called the Xcp secreton. The Xcp secreton shares many features with the machinery involved in type IV pilus assembly. More specifically, it involves the function of five pilin-like proteins, the XcpT-X pseudopilins. We show that, upon overexpression, the XcpT pseudopilin can be assembled in a pilus, which we call a type II pseudopilus. Image analysis and filtering of electron micrographs indicated that these appendages are composed of individual fibrils assembled together in a bundle structure. Our observations thus revealed that XcpT has properties similar to those of type IV pilin subunits. Interestingly, the assembly of the type II pseudopilus is not exclusively dependent on the Xcp machinery but can be supported by other similar machineries, such as the Pil (type IV pilus) and Hxc (type II secretion) systems of P. aeruginosa. In addition, heterologous pseudopilins can be assembled by P. aeruginosa into a type II pseudopilus. Finally, we showed that assembly of the type II pseudopilus confers increased bacterial adhesive capabilities. These observations confirmed the ability of pseudopilins to form a pilus structure and raise questions with respect to their function in terms of secretion and adhesion, two crucial biological processes in the course of bacterial infections.     

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. 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 Gln(29) 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.     

Structural characterization of protein secretion genes of the bacterial phytopathogen Xanthomonas campestris pathovar campestris: relatedness to secretion systems of other gram-negative bacteria

Summary The nucleotide sequence was determined of a 5.3 kb region of the Xanthomonas campestris pathovar campestris genome carrying a gene cluster encoding protein secretion and pathogenicity functions. A putative promoter sequence and five open reading frames (ORF) which may be part of an operon were revealed. The five predicted primary translation products comprise 531, 390, 147, 169 and 138 amino acids with M_r values of 58854, 42299, 15548, 18214 and 15108 respectively. A sixth, partial ORF is also present. Between ORF1 and ORF2 is a sequence of unknown function showing 7 by duplications. The deduced amino acid sequence of ORF1 is related to the Klebsiella pneumoniae PulE protein, to the Bacillus subtilis ComG ORF1 and to the Agrobacterium tumefaciens VirB ORF11 products. In addition, the deduced amino acid sequence of ORF2 showed homology to the Pu1F and to the ComG ORF2 products. The proteins encoded by ORF3, 4 and 5 showed amino acid homology to PulG, H and I products respectively. The proteins encoded by ORF2, 3, 4 and 5 showed significant hydrophobic domains which may represent membrane-spanning regions. By contrast the protein encoded by ORF1 was largely hydrophilic and had two putative nucleoside triphosphate binding sites.The nucleotide sequence data in this paper have been deposited in the EMBL, Genbank and DDBJ nucleotide sequence databases under the accession number X59079     

Assembly of the type II secretion machinery of Erwinia chrysanthemi: direct interaction and associated conformational change between OutE, the putative ATP-binding component and the membrane protein OutL.

Erwinia chrysanthemi secretes, by the type II secretory pathway, a large number of enzymes, including cellulases and pectinases. This process requires the products of the out genes, which are widely conserved in Gram-negative bacteria. The Out proteins are thought to form a membrane-associated multiprotein complex. Here, we investigated interaction between OutE, the putative ATP binding component, and OutL, an inner membrane protein. We showed, by limited proteolysis, genetic suppression and the yeast two-hybrid system, that OutE and OutL interact directly. Analysis of truncated forms of OutE demonstrated that the N terminus of OutE (residues 1-97) is important for the OutE/OutL interaction. Moreover, results from the yeast two-hybrid system suggested that OutE and OutL are each able to form homomultimers. The region required for homomultimerisation of OutE is located in its C terminus. Limited proteolysis assay indicated that OutE induces a conformational change in OutL, in both its cytoplasmic and periplasmic domains. Moreover, the secretion process requires a conformational change in OutE which depends on both the interaction with OutL and on the presence of an intact Walker A motif in OutE. Our results support the view that interaction occurring on the cytoplasmic side influences the events occurring in the outer membrane. We discuss a model in which OutE uses ATP to control the assembly of the type II secretion machinery.     

Gene integration and expression and extracellular secretion of Erwinia chrysanthemi endoglucanase CelY (celY) and CelZ (celZ) in ethanologenic Klebsiella oxytoca P2

The development of methods to reduce costs associated with the solubilization of cellulose is essential for the utilization of lignocellulose as a renewable feedstock for fuels and chemicals. One promising approach is the genetic engineering of ethanol-producing microorganisms that also produce cellulase enzymes during fermentation. By starting with an ethanologenic derivative (strain P2) of Klebsiella oxytoca M5A1 with the native ability to metabolize cellobiose, the need for supplemental beta-glucosidase was previously eliminated. In the current study, this approach has been extended by adding genes encoding endoglucanase activities. Genes celY and celZ from Erwinia chrysanthemi have been functionally integrated into the chromosome of P2 using surrogate promoters from Zymomonas mobilis for expression. Both were secreted into the extracellular milieu, producing more than 20,000 endoglucanase units (carboxymethyl cellulase activity) per liter of fermentation broth. During the fermentation of crystalline cellulose with low levels of commercial cellulases of fungal origin, these new strains produced up to 22% more ethanol than unmodified P2. Most of the beneficial contribution was attributed to CelY rather than to CelZ. These results suggest that fungal enzymes with substrate profiles resembling CelY (preference for long-chain polymers and lack of activity on soluble cello-oligosaccharides of two to five glucosyl residues) may be limiting in commercial cellulase preparations.     

A superfamily of proteins involved in different secretion pathways in gram-negative bacteria: modular structure and specificity of the N-terminal domain

The family of PulD proteins, which has been characterized in a wide variety of microorganisms, comprises several membrane-associated proteins essential for the transport of macromolecules across bacterial membranes. These proteins are involved in the transport of complex structures (such as phage particles, DNA) or various proteins (such as extracellular enzymes and pathogenicity determinants). Amino acid sequence analysis revealed a possible modular organisation of proteins of this superfamily, with highly conserved C-terminal domains and dissimilar N-terminal domains. In the C-terminal domain, four highly conserved regions have been found, one of them containing a remarkable common motif: (V, I)PXL(S, G)XIPXXGXLF. Structural comparisons between the N-terminal domains indicate that proteins of this superfamily can be divided into at least two subgroups, probably reflecting the existence of distinct secretion mechanisms. This implies that members of the superfamily of PulD-related proteins are independently involved in (1) the general secretory pathway, (2) a new signal-peptide-independent secretion pathway found in several bacterial pathogens, and possibly in (3) the translocation of bacteriophage particles through the bacterial cell envelope.     

Cloning and expression in Escherichia coli of the Serratia marcescens metalloprotease gene: secretion of the protease from E. coli in the presence of the Erwinia chrysanthemi protease secretion functions.

The Serratia marcescens extracellular protease SM is secreted by a signal peptide-independent pathway. When the prtSM gene was cloned and expressed in Escherichia coli, the cells did not secrete protease SM. The lack of secretion could be very efficiently complemented by the Erwinia chrysanthemi protease B secretion apparatus constituted by the PrtD, PrtE, and PrtF proteins. As with protease B and alpha-hemolysin, the secretion signal was located within the last 80 amino acids of the protease. These results indicate that the mechanism of S. marcescens protease SM secretion is analogous to the mechanisms of protease B and hemolysin secretion.     

Method for characterization of the enzyme profile and the determination of CBH I (Cel 7a) core protein in Trichoderma reesei cellulase preparations

Summary Fast protein liquid chromatography (FPLC) was used to characterize a commercial cellulase preparation (Celluclast 1.5L, Novozymes) in relation to its protein profile and activity against hydroxyethylcellulose (HEC) and other substrates. Co-elution of CBHII (Cel 6A) with other enzyme components of the cellulase system was characterized by immunochemical assays using monoclonal antibodies, whereas the occurrence of EGII (Cel 5A) was assessed based on its ability to cleave the heterosidic bond of 4-methylumbellyferyl-β-d-cellotrioside (MUmbG₃). The main cellulase constituents of Celluclast 1.5L were pooled into six fractions containing EGII (Cel 5A) and EGIII (Cel 12A) (F1), EGII and CBHII (Cel 6A) (F2), CBHII and EGI (Cel 7B) (F3), EGI (F4), and CBHI (Cel 7A) (F5). The occurrence of CBHI core protein within the CBHI fraction of the FPLC profile was determined by hydrophobic interaction chromatography. Using this method, we were able to demonstrate that the batch of Celluclast 1.5L used in this study contained 10.9–18.8% of CBHI as its corresponding free core protein.