scholarly journals New NodW- or NifA-Regulated Bradyrhizobium japonicum Genes

2003 ◽  
Vol 16 (4) ◽  
pp. 342-351 ◽  
Author(s):  
Isabelle Caldelari Baumberger ◽  
Nicole Fraefel ◽  
Michael Göttfert ◽  
Hauke Hennecke
Keyword(s):  
Bradyrhizobium Japonicum ◽  
Plant Cell Wall ◽  
Outer Membrane Proteins ◽  
Cellulase Activity ◽  
Dependent Manner ◽  
Cell Wall Degrading Enzymes ◽  
Reading Frame ◽  
Expression Studies ◽  
Lacz Fusions ◽  
In The Wild

A cluster of genes coding for putative plant cell-wall degrading enzymes (i.e., genes for two endoglucanases [gunA and gunA2], one pectinmethylesterase [pme], and one polygalacturonase [pgl]) was identified by sequence similarities in the symbiotic region of the Bradyrhizobium japonicum chromosome. In addition, a systematic screen of the region revealed several genes potentially transcribed by the σ54-RNA polymerase and activated by the transcriptional regulator NifA (i.e., genes for proteins with similarity to outer membrane proteins [id117 and id525] and a citrate carrier [id331 or citA] and one open reading frame without similarity to known proteins [id747]). Expression studies using transcriptional lacZ fusions showed that gunA2 and pgl were strongly induced by the isoflavone genistein in a NodW-dependent manner, suggesting a role of the gene products in early events of the nodulation process; by contrast, gunA and pme expression was very weak in the conditions tested. The gunA2 gene product was purified and was shown to have cellulase activity. β-Galactosidase activity expressed from transcriptional lacZ fusions to id117, id525, and id747 in the wild type and in nifA and rpoN mutant backgrounds confirmed that their transcription was dependent on NifA and σ54. Despite the presence of a -24/-12-type promoter and a NifA binding site upstream of citA, no regulation could be demonstrated in this case. Null mutations introduced in gunA, gunA2, pgl, pme, citA, id117, id525, and id747 did not impair the symbiosis with the host plants.

scholarly journals Cloning and expression of cellulase XF-818 of Xylella fastidiosa in Escherichia Coli

2003 ◽  
Vol 60 (4) ◽  
pp. 715-721 ◽  
Author(s):  
Nelson Arno Wulff ◽  
Helaine Carrer ◽  
Sérgio Florentino Pascholati
Keyword(s):  
Plant Pathogen ◽  
Plant Cell Wall ◽  
Heterologous Protein ◽  
Cellulase Activity ◽  
Comparative Sequence Analysis ◽  
Cloning And Expression ◽  
Heterologous Protein Expression ◽  
Cell Wall Degrading Enzymes ◽  
Endoglucanase Activity ◽  
Reading Frame

Xylella fastidiosa's genome was the first of a plant pathogen to be completely sequenced. Through comparative sequence analysis many genes were identified and, among them, several potentially involved in plant-pathogen interaction. However, the biological role of each gene should be assigned experimentally. On this regard, heterologous protein expression is a powerful tool to produce proteins from such genes, allowing their characterization. X. fastidiosa lives inside xylem vessels and eventually would degrade pit membranes from xylem cells to move radialy into the host. The identification of several putative plant cell wall degrading enzymes on X. fastidiosa genome prompted the assession of the function of such proteins. The open reading frame (ORF) Xf-818 was cloned into expression vector pET20b and E. coli cells harboring such plasmid exhibited cellulase activity. Using IPTG at 0.4 mmol L-1 with a 12 h incubation at 32°C are the best conditions to produce higher amounts of heterologous protein. The enzyme degrades cellulose confirming the endoglucanase activity of Xf-818.

scholarly journals Synergistic Effect of Different Plant Cell Wall–Degrading Enzymes Is Important for Virulence of Fusarium graminearum

2017 ◽  
Vol 30 (11) ◽  
pp. 886-895 ◽  
Author(s):  
Maria Chiara Paccanaro ◽  
Luca Sella ◽  
Carla Castiglioni ◽  
Francesca Giacomello ◽  
Ana Lilia Martínez-Rocha ◽  
...  
Keyword(s):  
Cell Wall ◽  
Fusarium Graminearum ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Xylanase Activity ◽  
Cellulase Activity ◽  
Wild Type ◽  
Cell Wall Degrading Enzymes ◽  
Xylanase Production ◽  
Significant Difference

Endo-polygalacturonases (PGs) and xylanases have been shown to play an important role during pathogenesis of some fungal pathogens of dicot plants, while their role in monocot pathogens is less defined. Pg1 and xyr1 genes of the wheat pathogen Fusarium graminearum encode the main PG and the major regulator of xylanase production, respectively. Single- and double-disrupted mutants for these genes were obtained to assess their contribution to fungal infection. Compared with wild-type strain, the ∆pg mutant showed a nearly abolished PG activity, slight reduced virulence on soybean seedlings, but no significant difference in disease symptoms on wheat spikes; the ∆xyr mutant was strongly reduced in xylanase activity and moderately reduced in cellulase activity but was as virulent as wild type on both soybean and wheat plants. Consequently, the ΔpgΔxyr double mutant was impaired in xylanase, PG, and cellulase activities but, differently from single mutants, was significantly reduced in virulence on both plants. These findings demonstrate that the concurrent presence of PG, xylanase, and cellulase activities is necessary for full virulence. The observation that the uronides released from wheat cell wall after a F. graminearum PG treatment were largely increased by the fungal xylanases suggests that these enzymes act synergistically in deconstructing the plant cell wall.

scholarly journals Developing a method for real-time visualization of cellulase activity

2020 ◽  
Author(s):  
Pallavi Kumari ◽  
Tali Sayas ◽  
Patricia Bucki ◽  
Sigal Brown Miyara ◽  
Maya Kleiman
Keyword(s):  
Real Time ◽  
Plant Cell Wall ◽  
Plant Root ◽  
Cellulase Activity ◽  
Root Surface ◽  
Surface Microstructure ◽  
Root Extract ◽  
Cell Wall Degrading Enzymes ◽  
Real Time Visualization ◽  
Replication Technique

AbstractStudying the interactions between microorganisms and plant roots is crucial for understanding a variety of phenomena concerning crop yield and health. The role of root surface properties in these interactions, is rarely addressed. To this end, we previously built a synthetic system, from the inert polymer polydimethyl siloxane (PDMS), mimicking the root surface microstructure, using a replication technique. This replica enables the study of isolated effects of surface structure on microorganism-plant interactions. Since the root surface is composed mostly of cellulose, using cellulose-like materials as our replica, instead of PDMS, is the next logical step. This will enable following the hydrolysis of such surfaces as a result of microorganisms secreting Plant Cell Wall Degrading Enzymes (PCWDE), and in particular, cellulase. Visualization of such hydrolysis in a synthetic system can assist in studying the localization and activity of microorganisms and how they correlate with surface microtopography, separately from chemical plant signals.In this work, we modified the known carboxymethyl cellulase (CMC) hydrolysis visualization method to enable real-time tracking of cellulase activity of microorganisms on the surface. Surface was formed from pure CMC, rather than CMC incorporated in agar as is often done, and by that, eliminating diffusion issues. Acridine orange dye, which is compatible, at low concentrations, with microorganisms, as opposed to other routinely used dyes, was incorporated into the film. The dye disassociated from the film when hydrolysis occurred, forming a halo surrounding the point of hydrolysis. This enabled real-time visualization since the common need for post hydrolysis dyeing was negated. Using Root Knot Nematode (RKN) as a model organism that penetrates the plant root, we showed it was possible to follow microorganism cellulase secretion on the surface in the form of CMC film hydrolysis. Furthermore, the addition of natural additives, in the form of root extract was also shown to be an option and resulted in an increased RKN response. We tested our newly developed method by changing temperature and pH conditions and by characterization of the hydrolyzed surface using both Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM).This method will be implemented in the future on a root surface microstructure replica. We believe the combination of this new method with our previously developed root surface microstructure replication technique can open a new avenue of research in the field of plant root-microorganism interactions.

scholarly journals Iron-Dependent Cytochrome c1 Expression Is Mediated by the Status of Heme in Bradyrhizobium japonicum

2005 ◽  
Vol 187 (15) ◽  
pp. 5084-5089 ◽  
Author(s):  
Tao Gao ◽  
Mark R. O'Brian
Keyword(s):  
Bradyrhizobium Japonicum ◽  
Aminolevulinic Acid ◽  
Iron Status ◽  
Covalent Binding ◽  
Dependent Manner ◽  
Wild Type ◽  
Prosthetic Group ◽  
Protein Levels ◽  
Genes Encoding ◽  
In The Wild

ABSTRACT The heme prosthetic group of heme proteins contains iron, which can be a limiting nutrient. Here, we show that cytochrome c 1 protein from Bradyrhizobium japonicum was strongly affected by the iron status, with low expression in cells grown under iron limitation. This control was not affected in mutants encoding the iron regulator Irr or Fur. Furthermore, cytochrome c 1 mRNA was not influenced by the iron status, suggesting control at a posttranscriptional step. Cytochrome c 1 protein levels were very low in mutants defective in the genes encoding δ-aminolevulinic acid (ALA) synthase and ferrochelatase, enzymes that catalyze the first and final steps of the heme biosynthetic pathway, respectively. Iron-dependent cytochrome c 1 expression was restored in the ALA synthase mutant by supplementation of the medium with the heme precursor ALA. Supplementation with heme resulted in high levels of cytochrome c 1 protein in the wild type and in both mutants, but expression was no longer iron dependent. Cytochrome c 1 is synthesized as a protein precursor fused with cytochrome b. A plasmid-borne construct encoding only cytochrome c 1 was expressed in an iron- and heme-dependent manner similar to that of the wild-type gene, indicating that control by those effectors is not linked to posttranslational processing of the fusion protein. Mutation of the cytochrome c 1 cysteines involved in covalent binding to heme nearly abolished immunodetectable protein. Thus, defects in heme synthesis or heme binding abrogate cytochrome c 1 accumulation, apparently due to protein degradation. We suggest that iron-dependent cytochrome c 1 expression is mediated by heme availability for heme protein formation

scholarly journals Quorum sensing, virulence and secondary metabolite production in plant soft-rotting bacteria

2007 ◽  
Vol 362 (1483) ◽  
pp. 1165-1183 ◽  
Author(s):  
Anne M.L Barnard ◽  
Steven D Bowden ◽  
Tom Burr ◽  
Sarah J Coulthurst ◽  
Rita E Monson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Quorum Sensing ◽  
Plant Cell Wall ◽  
Antibiotic Production ◽  
Dependent Manner ◽  
Virulence Determinants ◽  
Cell Wall Degrading Enzymes ◽  
Acylhomoserine Lactone ◽  
Autoinducer 2 ◽  
Regulatory Systems

Quorum sensing describes the ability of bacteria to sense their population density and respond by modulating gene expression. In the plant soft-rotting bacteria, such as Erwinia , an arsenal of plant cell wall-degrading enzymes is produced in a cell density-dependent manner, which causes maceration of plant tissue. However, quorum sensing is central not only to controlling the production of such destructive enzymes, but also to the control of a number of other virulence determinants and secondary metabolites. Erwinia synthesizes both N -acylhomoserine lactone (AHL) and autoinducer-2 types of quorum sensing signal, which both play a role in regulating gene expression in the phytopathogen. We review the models for AHL-based regulation of carbapenem antibiotic production in Erwinia . We also discuss the importance of quorum sensing in the production and secretion of virulence determinants by Erwinia , and its interplay with other regulatory systems.

scholarly journals Distinct roles of N- and O-glycans in cellulase activity and stability

2017 ◽  
Vol 114 (52) ◽  
pp. 13667-13672 ◽  
Author(s):  
Antonella Amore ◽  
Brandon C. Knott ◽  
Nitin T. Supekar ◽  
Asif Shajahan ◽  
Parastoo Azadi ◽  
...  
Keyword(s):  
Plant Cell Wall ◽  
Catalytic Domain ◽  
Domain Architecture ◽  
Cellulase Activity ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Cell Wall Degrading Enzymes ◽  
Cellulose Conversion ◽  
Degrading Enzymes

In nature, many microbes secrete mixtures of glycoside hydrolases, oxidoreductases, and accessory enzymes to deconstruct polysaccharides and lignin in plants. These enzymes are often decorated with N- and O-glycosylation, the roles of which have been broadly attributed to protection from proteolysis, as the extracellular milieu is an aggressive environment. Glycosylation has been shown to sometimes affect activity, but these effects are not fully understood. Here, we examine N- and O-glycosylation on a model, multimodular glycoside hydrolase family 7 cellobiohydrolase (Cel7A), which exhibits an O-glycosylated carbohydrate-binding module (CBM) and an O-glycosylated linker connected to an N- and O-glycosylated catalytic domain (CD)—a domain architecture common to many biomass-degrading enzymes. We report consensus maps for Cel7A glycosylation that include glycan sites and motifs. Additionally, we examine the roles of glycans on activity, substrate binding, and thermal and proteolytic stability. N-glycan knockouts on the CD demonstrate that N-glycosylation has little impact on cellulose conversion or binding, but does have major stability impacts. O-glycans on the CBM have little impact on binding, proteolysis, or activity in the whole-enzyme context. However, linker O-glycans greatly impact cellulose conversion via their contribution to proteolysis resistance. Molecular simulations predict an additional role for linker O-glycans, namely that they are responsible for maintaining separation between ordered domains when Cel7A is engaged on cellulose, as models predict α-helix formation and decreased cellulose interaction for the nonglycosylated linker. Overall, this study reveals key roles for N- and O-glycosylation that are likely broadly applicable to other plant cell-wall–degrading enzymes.

scholarly journals The Cloning and Molecular Analysis of pawn-B in Paramecium tetraurelia

Genetics ◽  
2000 ◽  
Vol 155 (3) ◽  
pp. 1105-1117 ◽  
Author(s):  
W John Haynes ◽  
Kit-Yin Ling ◽  
Robin R Preston ◽  
Yoshiro Saimi ◽  
Ching Kung
Keyword(s):  
Genomic Library ◽  
Open Reading Frame ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Rt Pcr ◽  
Reading Frame ◽  
Close Proximity ◽  
In The Wild ◽  
Dna Fragment ◽  
Alternative Sites

Abstract Pawn mutants of Paramecium tetraurelia lack a depolarization-activated Ca2+ current and do not swim backward. Using the method of microinjection and sorting a genomic library, we have cloned a DNA fragment that complements pawn-B (pwB/pwB). The minimal complementing fragment is a 798-bp open reading frame (ORF) that restores the Ca2+ current and the backward swimming when expressed. This ORF contains a 29-bp intron and is transcribed and translated. The translated product has two putative transmembrane domains but no clear matches in current databases. Mutations in the available pwB alleles were found within this ORF. The d4-95 and d4-96 alleles are single base substitutions, while d4-662 (previously pawn-D) harbors a 44-bp insertion that matches an internal eliminated sequence (IES) found in the wild-type germline DNA except for a single C-to-T transition. Northern hybridizations and RT-PCR indicate that d4-662 transcripts are rapidly degraded or not produced. A second 155-bp IES in the wild-type germline ORF excises at two alternative sites spanning three asparagine codons. The pwB ORF appears to be separated from a 5′ neighboring ORF by only 36 bp. The close proximity of the two ORFs and the location of the pwB protein as indicated by GFP-fusion constructs are discussed.

scholarly journals Arabinanase A from Pseudomonas fluorescens subsp. cellulosa exhibits both an endo- and an exo- mode of action

1997 ◽  
Vol 323 (2) ◽  
pp. 547-555 ◽  
Author(s):  
Vincent A. McKIE ◽  
Gary W. BLACK ◽  
Sarah J. MILLWARD-SADLER ◽  
Geoffrey P. HAZLEWOOD ◽  
Judith I. LAURIE ◽  
...  
Keyword(s):  
Pseudomonas Fluorescens ◽  
Mode Of Action ◽  
Plant Cell Wall ◽  
Catalytic Domain ◽  
Genomic Library ◽  
Glycosyl Hydrolase ◽  
Single Copy ◽  
Terminal Sequence ◽  
Reading Frame ◽  
Significant Release

Pseudomonas fluorescens subsp. cellulosa expressed arabinanase activity when grown on media supplemented with arabinan or arabinose. Arabinanase activity was not induced by the inclusion of other plant structural polysaccharides, and was repressed by the addition of glucose. The majority of the Pseudomonas arabinanase activity was extracellular. Screening of a genomic library of P. fluorescens subsp. cellulosa DNA constructed in Lambda ZAPII, for recombinants that hydrolysed Red-dyed arabinan, identified five arabinan-degrading plaques. Each of the phage contained the same Pseudomonas arabinanase gene, designated arbA, which was present as a single copy in the Pseudomonas genome. The nucleotide sequence of arbA revealed an open reading frame of 1041 bp encoding a protein, designated arabinanase A (ArbA), of Mr 39438. The N-terminal sequence of ArbA exhibited features typical of a prokaryotic signal peptide. Analysis of the primary structure of ArbA indicated that, unlike most Pseudomonas plant cell wall hydrolases, it did not contain linker sequences or have a modular structure, but consisted of a single catalytic domain. Sequence comparison between the Pseudomonas arabinanase and proteins in the SWISS-PROT database showed that ArbA exhibits greatest sequence identity with arabinanase A from Aspergillus niger, placing the enzyme in glycosyl hydrolase Family 43. The significance of the differing substrate specificities of enzymes in Family 43 is discussed. ArbA purifed from a recombinant strain of Escherichia coli had an Mr of 34000 and an N-terminal sequence identical to residues 32–51 of the deduced sequence of ArbA, and hydrolysed linear arabinan, carboxymethylarabinan and arabino-oligosaccharides. The enzyme displayed no activity against other plant structural polysaccharides, including branched sugar beet arabinan. ArbA produced almost exclusively arabinotriose from linear arabinan and appeared to hydrolyse arabino-oligosaccharides by successively releasing arabinotriose. ArbA and the Aspergillus arabinanase mediated a decrease in the viscosity of linear arabinan that was associated with a significant release of reducing sugar. We propose that ArbA is an arabinanase that exhibits both an endo- and an exo- mode of action.

An electrogenic Na+- HCO 3 − cotransporter (NBC) with a novel COOH-terminus, cloned from rat brain

2000 ◽  
Vol 278 (6) ◽  
pp. C1200-C1211 ◽  
Author(s):  
Mark O. Bevensee ◽  
Bernhard M. Schmitt ◽  
Inyeong Choi ◽  
Michael F. Romero ◽  
Walter F. Boron
Keyword(s):  
Rat Brain ◽  
Cortical Neurons ◽  
Polyclonal Antibodies ◽  
Amino Acid Level ◽  
Cdna Libraries ◽  
Human Pancreas ◽  
Opposite Pattern ◽  
Reading Frame ◽  
Expression Studies ◽  
Rapid Amplification

We screened rat brain cDNA libraries and used 5′ rapid amplification of cDNA ends to clone two electrogenic Na+-[Formula: see text] cotransporter (NBC) isoforms from rat brain (rb1NBC and rb2NBC). At the amino acid level, one clone (rb1NBC) is 96% identical to human pancreas NBC. The other clone (rb2NBC) is identical to rb1NBC except for 61 unique COOH-terminal amino acids, the result of a 97-bp deletion near the 3′ end of the open-reading frame. Using RT-PCR, we confirmed that mRNA from rat brain contains this 97-bp deletion. Furthermore, we generated rabbit polyclonal antibodies that distinguish between the unique COOH-termini of rb1NBC (αrb1NBC) and rb2NBC (αrb2NBC). αrb1NBC labels an ∼130-kDa protein predominantly from kidney, and αrb2NBC labels an ∼130-kDa protein predominantly from brain. αrb2NBC labels a protein that is more highly expressed in cortical neurons than astrocytes cultured from rat brain; αrb1NBC exhibits the opposite pattern. In expression studies, applying 1.5% CO2/10 mM [Formula: see text] to Xenopus oocytes injected with rb2NBC cRNA causes 1) pHi to recover from the initial CO2-induced acidification and 2) the cell to hyperpolarize. Subsequently, removing external Na+ reverses the pHi increase and elicits a rapid depolarization. In the presence of 450 μM DIDS, removing external Na+ has no effect on pHi and elicits a small hyperpolarization. The rate of the pHidecrease elicited by removing Na+ is insensitive to removing external Cl−. Thus rb2NBC is a DIDS-sensitive, electrogenic NBC that is predominantly expressed in brain of at least rat.

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