scholarly journals Gene Integration and Expression and Extracellular Secretion of Erwinia chrysanthemi Endoglucanase CelY (celY) and CelZ (celZ) in EthanologenicKlebsiella oxytoca P2

2001 ◽  
Vol 67 (1) ◽  
pp. 6-14 ◽  
Author(s):  
Shengde Zhou ◽  
F. C. Davis ◽  
L. O. Ingram
Keyword(s):  
Fermentation Broth ◽  
Klebsiella Oxytoca ◽  
Cellulase Activity ◽  
Erwinia Chrysanthemi ◽  
Crystalline Cellulose ◽  
Fungal Enzymes ◽  
Extracellular Milieu ◽  
Extracellular Secretion ◽  
Genes Encoding ◽  
Fuels And Chemicals

ABSTRACT 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) ofKlebsiella oxytoca M5A1 with the native ability to metabolize cellobiose, the need for supplemental β-glucosidase was previously eliminated. In the current study, this approach has been extended by adding genes encoding endoglucanase activities. GenescelY 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.

scholarly journals A Metzincin and TIMP-Like Protein Pair of a Phage Origin Sensitize Listeria monocytogenes to Phage Lysins and Other Cell Wall Targeting Agents

2021 ◽  
Vol 9 (6) ◽  
pp. 1323
Author(s):  
Etai Boichis ◽  
Nadejda Sigal ◽  
Ilya Borovok ◽  
Anat A. Herskovits
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Mammalian Cells ◽  
Protein Pair ◽  
Growth Conditions ◽  
Wall Structure ◽  
Antibiotic Resistant ◽  
Extracellular Milieu ◽  
Virion Release ◽  
Genes Encoding

Infection of mammalian cells by Listeria monocytogenes (Lm) was shown to be facilitated by its phage elements. In a search for additional phage remnants that play a role in Lm’s lifecycle, we identified a conserved locus containing two XRE regulators and a pair of genes encoding a secreted metzincin protease and a lipoprotein structurally similar to a TIMP-family metzincin inhibitor. We found that the XRE regulators act as a classic CI/Cro regulatory switch that regulates the expression of the metzincin and TIMP-like genes under intracellular growth conditions. We established that when these genes are expressed, their products alter Lm morphology and increase its sensitivity to phage mediated lysis, thereby enhancing virion release. Expression of these proteins also sensitized the bacteria to cell wall targeting compounds, implying that they modulate the cell wall structure. Our data indicate that these effects are mediated by the cleavage of the TIMP-like protein by the metzincin, and its subsequent release to the extracellular milieu. While the importance of this locus to Lm pathogenicity remains unclear, the observation that this phage-associated protein pair act upon the bacterial cell wall may hold promise in the field of antibiotic potentiation to combat antibiotic resistant bacterial pathogens.

scholarly journals Synergistic Hydrolysis of Carboxymethyl Cellulose and Acid-Swollen Cellulose by Two Endoglucanases (CelZ and CelY) fromErwinia chrysanthemi

2000 ◽  
Vol 182 (20) ◽  
pp. 5676-5682 ◽  
Author(s):  
Shengde Zhou ◽  
Lonnie O. Ingram
Keyword(s):  
Carboxymethyl Cellulose ◽  
Cell Walls ◽  
Cellulase Activity ◽  
Substrate Preference ◽  
Erwinia Chrysanthemi ◽  
General Mechanism ◽  
Plant Cell Walls ◽  
Enzymatic Modification ◽  
Amorphous Cellulose ◽  
Hydrolysis Of

ABSTRACT Erwinia chrysanthemi produces a battery of hydrolases and lyases which are very effective in the maceration of plant cell walls. Although two endoglucanases (CelZ and CelY; formerly EGZ and EGY) are produced, CelZ represents approximately 95% of the total carboxymethyl cellulase activity. In this study, we have examined the effectiveness of CelY and CelZ alone and of combinations of both enzymes using carboxymethyl cellulose (CMC) and amorphous cellulose (acid-swollen cellulose) as substrates. Synergy was observed with both substrates. Maximal synergy (1.8-fold) was observed for combinations containing primarily CelZ; the ratio of enzyme activities produced was similar to those produced by cultures of E. chrysanthemi. CelY and CelZ were quite different in substrate preference. CelY was unable to hydrolyze soluble cellooligosaccharides (cellotetraose and cellopentaose) but hydrolyzed CMC to fragments averaging 10.7 glucosyl units. In contrast, CelZ readily hydrolyzed cellotetraose, cellopentaose, and amorphous cellulose to produce cellobiose and cellotriose as dominant products. CelZ hydrolyzed CMC to fragments averaging 3.6 glucosyl units. In combination, CelZ and CelY hydrolyzed CMC to products averaging 2.3 glucosyl units. Synergy did not require the simultaneous presence of both enzymes. Enzymatic modification of the substrate by CelY increased the rate and extent of hydrolysis by CelZ. Full synergy was retained by the sequential hydrolysis of CMC, provided CelY was used as the first enzyme. A general mechanism is proposed to explain the synergy between these two enzymes based primarily on differences in substrate preference.

scholarly journals Lack of a surface layer in Tannerella forsythia mutants deficient in the type IX secretion system

Microbiology ◽  
2014 ◽  
Vol 160 (10) ◽  
pp. 2295-2303 ◽  
Author(s):  
Yuka Narita ◽  
Keiko Sato ◽  
Hideharu Yukitake ◽  
Mikio Shoji ◽  
Daisuke Nakane ◽  
...  
Keyword(s):  
Surface Layer ◽  
Biofilm Formation ◽  
Anaerobic Bacterium ◽  
Secretion System ◽  
Tannerella Forsythia ◽  
Gram Negative ◽  
Extracellular Milieu ◽  
Genes Encoding ◽  
Type Ix Secretion System ◽  
Encoding Genes

Tannerella forsythia, a Gram-negative anaerobic bacterium, is an important pathogen in periodontal disease. This bacterium possesses genes encoding all known components of the type IX secretion system (T9SS). T. forsythia mutants deficient in genes orthologous to the T9SS-encoding genes porK, porT and sov were constructed. All porK, porT and sov single mutants lacked the surface layer (S-layer) and expressed less-glycosylated versions of the S-layer glycoproteins TfsA and TfsB. In addition, these mutants exhibited decreased haemagglutination and increased biofilm formation. Comparison of the proteins secreted by the porK and WT strains revealed that the secretion of several proteins containing C-terminal domain (CTD)-like sequences is dependent on the porK gene. These results indicate that the T9SS is functional in T. forsythia and contributes to the translocation of CTD proteins to the cell surface or into the extracellular milieu.

scholarly journals Genome Sequence of the Cellulolytic Gliding Bacterium Cytophaga hutchinsonii

2007 ◽  
Vol 73 (11) ◽  
pp. 3536-3546 ◽  
Author(s):  
Gary Xie ◽  
David C. Bruce ◽  
Jean F. Challacombe ◽  
Olga Chertkov ◽  
John C. Detter ◽  
...  
Keyword(s):  
Gliding Motility ◽  
Open Reading Frames ◽  
Crystalline Cellulose ◽  
Cellulolytic Bacteria ◽  
Cytophaga Hutchinsonii ◽  
Type Iv ◽  
Flavobacterium Johnsoniae ◽  
Anchoring Proteins ◽  
Genes Encoding ◽  
Gliding Bacterium

ABSTRACT The complete DNA sequence of the aerobic cellulolytic soil bacterium Cytophaga hutchinsonii, which belongs to the phylum Bacteroidetes, is presented. The genome consists of a single, circular, 4.43-Mb chromosome containing 3,790 open reading frames, 1,986 of which have been assigned a tentative function. Two of the most striking characteristics of C. hutchinsonii are its rapid gliding motility over surfaces and its contact-dependent digestion of crystalline cellulose. The mechanism of C. hutchinsonii motility is not known, but its genome contains homologs for each of the gld genes that are required for gliding of the distantly related bacteroidete Flavobacterium johnsoniae. Cytophaga-Flavobacterium gliding appears to be novel and does not involve well-studied motility organelles such as flagella or type IV pili. Many genes thought to encode proteins involved in cellulose utilization were identified. These include candidate endo-β-1,4-glucanases and β-glucosidases. Surprisingly, obvious homologs of known cellobiohydrolases were not detected. Since such enzymes are needed for efficient cellulose digestion by well-studied cellulolytic bacteria, C. hutchinsonii either has novel cellobiohydrolases or has an unusual method of cellulose utilization. Genes encoding proteins with cohesin domains, which are characteristic of cellulosomes, were absent, but many proteins predicted to be involved in polysaccharide utilization had putative D5 domains, which are thought to be involved in anchoring proteins to the cell surface.

scholarly journals Iron Regulation and Pathogenicity in Erwinia chrysanthemi 3937: Role of the Fur Repressor Protein

1999 ◽  
Vol 12 (2) ◽  
pp. 119-128 ◽  
Author(s):  
Thierry Franza ◽  
Christele Sauvage ◽  
Dominique Expert
Keyword(s):  
Iron Transport ◽  
Reverse Genetics ◽  
Erwinia Chrysanthemi ◽  
Transport Systems ◽  
Iron Regulation ◽  
Iron Availability ◽  
Reading Frame ◽  
Genes Encoding ◽  
Coordinated Expression ◽  
Main Determinants

Low iron availability is a triggering signal for coordinated expression of the genes encoding pectate lyases PelB, PelC, PelD, and PelE, and chrysobactin iron transport functions, which are two main determinants of phytopathogenicity of the Erwinia chrysanthemi strain 3937. The possible implication of the ferric uptake regulation (Fur) protein in this process was investigated. The E. chrysanthemi fur gene was cloned by functional complementation of an Escherichia coli fur mutant and sequenced. The 444-bp open reading frame identified was found to code for a protein highly similar to the E. coli Fur regulator. An E. chrysanthemi fur null mutant was constructed by reverse genetics. This mutant showed altered growth capacity and reduced pathogenicity on African violets. In a fur background, transcriptional lacZ fusions to genes belonging to the E. chrysanthemi high affinity iron transport systems were constitutively expressed. Transcription of the pelA, pelD, and pelE genes was analyzed, using fusions to the uidA reporter gene. Iron availability and a fur mutation did not influence the expression of pelA. In the presence of iron, pelD and pelE transcription levels were higher in the fur mutant than in the parental strain. Furthermore, iron deficiency stimulated the expression of both fusions in the fur mutant. These findings indicate that, in E. chrysanthemi 3937, (i) Fur negatively controls iron transport and genes encoding PelD and PelE, and (ii) additional factor(s) mediate iron regulation of the pel genes.

scholarly journals Enzyme Diversity of the Cellulolytic System Produced by Clostridium cellulolyticum Explored by Two-Dimensional Analysis: Identification of Seven Genes Encoding New Dockerin-Containing Proteins

2007 ◽  
Vol 189 (6) ◽  
pp. 2300-2309 ◽  
Author(s):  
Jean-Charles Blouzard ◽  
Caroline Bourgeois ◽  
Pascale de Philip ◽  
Odile Valette ◽  
Anne Bélaïch ◽  
...  
Keyword(s):  
Energy Source ◽  
Glycoside Hydrolase ◽  
Glycoside Hydrolases ◽  
Crystalline Cellulose ◽  
Two Dimensional ◽  
Specific Antibodies ◽  
Two Dimensional Electrophoresis ◽  
Clostridium Cellulolyticum ◽  
Genes Encoding ◽  
Dimensional Electrophoresis

ABSTRACT The enzyme diversity of the cellulolytic system produced by Clostridium cellulolyticum grown on crystalline cellulose as a sole carbon and energy source was explored by two-dimensional electrophoresis. The cellulolytic system of C. cellulolyticum is composed of at least 30 dockerin-containing proteins (designated cellulosomal proteins) and 30 noncellulosomal components. Most of the known cellulosomal proteins, including CipC, Cel48F, Cel8C, Cel9G, Cel9E, Man5K, Cel9M, and Cel5A, were identified by using two-dimensional Western blot analysis with specific antibodies, whereas Cel5N, Cel9J, and Cel44O were identified by using N-terminal sequencing. Unknown enzymes having carboxymethyl cellulase or xylanase activities were detected by zymogram analysis of two-dimensional gels. Some of these enzymes were identified by N-terminal sequencing as homologs of proteins listed in the NCBI database. Using Trap-Dock PCR and DNA walking, seven genes encoding new dockerin-containing proteins were cloned and sequenced. Some of these genes are clustered. Enzymes encoded by these genes belong to glycoside hydrolase families GH2, GH9, GH10, GH26, GH27, and GH59. Except for members of family GH9, which contains only cellulases, the new modular glycoside hydrolases discovered in this work could be involved in the degradation of different hemicellulosic substrates, such as xylan or galactomannan.

scholarly journals XpsG, the major pseudopilin in Xanthomonas campestris pv. campestris, forms a pilus-like structure between cytoplasmic and outer membranes

2002 ◽  
Vol 365 (1) ◽  
pp. 205-211 ◽  
Author(s):  
Nien-Tai HU ◽  
Wei-Ming LEU ◽  
Meng-Shiunn LEE ◽  
Avon CHEN ◽  
Shu-Chung CHEN ◽  
...  
Keyword(s):  
Xanthomonas Campestris ◽  
Klebsiella Oxytoca ◽  
Subcellular Fractionation ◽  
Soluble Form ◽  
Wild Type ◽  
Outer Membranes ◽  
Extracellular Milieu ◽  
Large Complex ◽  
Xanthomonas Campestris Pv Campestris

GspG, -H, -I, -J and -K proteins are members of the pseudopilin family. They are the components required for the type II secretion pathway, which translocates proteins across the outer membrane of Gram-negative bacteria to the extracellular milieu. They were predicted to form a pilus-like structure, and this has been shown for PulG of Klebsiella oxytoca by using electron microscopy. In the present study, we performed biochemical analyses of the XpsG protein of Xanthomonas campestris pv. campestris and observed that it is a pillar-like structure spanning the cytoplasmic and outer membranes. Subcellular fractionation revealed a soluble form (SF) of XpsG, in addition to the membrane form. Chromatographic analysis of SF XpsG in the absence of a detergent indicated that it is part of a large complex (>440kDa). In vitro studies indicated that XpsG is prone to aggregate in the absence of a detergent. We isolated and characterized a non-functional mutant defective in forming the large complex. It did not interfere with the function of wild-type XpsG and was not detectable in the SF. Moreover, unlike wild-type XpsG, which was distributed in both the cytoplasmic and outer membranes, it appeared only in the cytoplasmic membrane. When wild-type XpsG was co-expressed with His6-tagged XpsH but not with untagged XpsH, SF XpsG bound to nickel and co-eluted with XpsH. This result suggests the presence of other pseudopilin components in the XpsG-containing large-sized molecules.

scholarly journals Coupling of Iron Assimilation and Pectinolysis in Erwinia chrysanthemi 3937

2002 ◽  
Vol 15 (11) ◽  
pp. 1181-1191 ◽  
Author(s):  
Thierry Franza ◽  
Isabelle Michaud-Soret ◽  
Pierrette Piquerel ◽  
Dominique Expert
Keyword(s):  
Iron Transport ◽  
Molecular Mechanisms ◽  
Constitutive Expression ◽  
Erwinia Chrysanthemi ◽  
Activation Mechanism ◽  
Receptor Protein ◽  
Virulence Determinants ◽  
Pectate Lyases ◽  
Iron Assimilation ◽  
Genes Encoding

Two major virulence determinants of the plant-pathogenic enterobacterium Erwinia chrysanthemi strain 3937 are the production of pectate lyase enzymes that degrade plant cell walls and expression of two high-affinity iron uptake systems mediated by two structurally unrelated siderophores, chrysobactin and achromobactin. Low iron availability is a signal that triggers transcription of the genes encoding pectate lyases PelD and PelE as well as that of genes involved in iron transport. This metalloregulation is mediated by the transcriptional repressor Fur. In this study, we analyzed the molecular mechanisms of this control. We purified the Erwinia chrysanthemi Fur protein. Band shift assays showed that Fur specifically binds in vitro to the regulatory regions of the genes encoding the ferrichrysobactin outer membrane receptor Fct and the pectate lyases PelD and PelE. We identified the Fur-binding sites of these promoter regions by performing DNase I footprinting experiments. From these data, we propose that Fur could inhibit the activation of the pelD and pelE genes by the cAMP receptor protein CRP according to an anti-activation mechanism. To identify other possible effectors involved in this control, we screened a bank of insertion mutants for an increase in transcriptional activity of pelD and fct genes in response to iron limitation. We isolated a mutant affected in the kdgK gene encoding the 2-keto-3-deoxygluconate (KDG) kinase, an enzyme involved in pectin catabolism. The growth of this mutant in the presence of pectic compounds led to a constitutive expression of iron transport genes as well as complete derepression of the pectinolysis genes. This effect was caused by intracellular accumulation of KDG. However, the derepression of iron transport genes by KDG does not involve the KdgR regulator of pectinolysis genes, which uses KDG as inducer. Thus, in Erwinia chrysanthemi, iron depletion or presence of KDG induces transcription of the genes involved in iron assimilation and pectinolysis. These important pathogenicity functions are coregulated by responding to common signals encountered in planta.

scholarly journals Characterization of the Erwinia chrysanthemi gan Locus, Involved in Galactan Catabolism

2007 ◽  
Vol 189 (19) ◽  
pp. 7053-7061 ◽  
Author(s):  
Aurélie Delangle ◽  
Anne-France Prouvost ◽  
Virginie Cogez ◽  
Jean-Pierre Bohin ◽  
Jean-Marie Lacroix ◽  
...  
Keyword(s):  
Abc Transporter ◽  
Transport System ◽  
Pathogenic Bacteria ◽  
Erwinia Chrysanthemi ◽  
Potato Tubers ◽  
Inner Membrane ◽  
Saintpaulia Ionantha ◽  
Genes Encoding

ABSTRACT β-1,4-Galactan is a major component of the ramified regions of pectin. Analysis of the genome of the plant pathogenic bacteria Erwinia chrysanthemi revealed the presence of a cluster of eight genes encoding proteins potentially involved in galactan utilization. The predicted transport system would comprise a specific porin GanL and an ABC transporter made of four proteins, GanFGK2. Degradation of galactans would be catalyzed by the periplasmic 1,4-β-endogalactanase GanA, which released oligogalactans from trimer to hexamer. After their transport through the inner membrane, oligogalactans would be degraded into galactose by the cytoplasmic 1,4-β-exogalactanase GanB. Mutants affected for the porin or endogalactanase were unable to grow on galactans, but they grew on galactose and on a mixture of galactotriose, galactotetraose, galactopentaose, and galactohexaose. Mutants affected for the periplasmic galactan binding protein, the transporter ATPase, or the exogalactanase were only able to grow on galactose. Thus, the phenotypes of these mutants confirmed the functionality of the gan locus in transport and catabolism of galactans. These mutations did not affect the virulence of E. chrysanthemi on chicory leaves, potato tubers, or Saintpaulia ionantha, suggesting an accessory role of galactan utilization in the bacterial pathogeny.

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