scholarly journals Novel Family of Carbohydrate-Binding Modules Revealed by the Genome Sequence of Spirochaeta thermophila DSM 6192

2011 ◽  
Vol 77 (15) ◽  
pp. 5483-5489 ◽  
Author(s):  
Angel Angelov ◽  
Christoph Loderer ◽  
Susanne Pompei ◽  
Wolfgang Liebl
Keyword(s):  
Genome Sequence ◽  
Sequence Data ◽  
Cellulose Degradation ◽  
Sequence Similarity ◽  
Open Reading Frames ◽  
Carbohydrate Binding ◽  
Functional Screening ◽  
Content Type ◽  
C Terminus ◽  
Carbohydrate Binding Modules

ABSTRACTSpirochaeta thermophilais a thermophilic, free-living, and cellulolytic anaerobe. The genome sequence data for this organism have revealed a high density of genes encoding enzymes from more than 30 glycoside hydrolase (GH) families and a noncellulosomal enzyme system for (hemi)cellulose degradation. Functional screening of a fosmid library whose inserts were mapped on theS. thermophilagenome sequence allowed the functional annotation of numerous GH open reading frames (ORFs). Seven different GH ORFs from theS. thermophilaDSM 6192 genome, all putative β-glycanase ORFs according to sequence similarity analysis, contained a highly conserved novel GH-associated module of unknown function at their C terminus. Four of these GH enzymes were experimentally verified as xylanase, β-glucanase, β-glucanase/carboxymethylcellulase (CMCase), and CMCase. Binding experiments performed with the recombinantly expressed and purified GH-associated module showed that it represents a new carbohydrate-binding module (CBM) that binds to microcrystalline cellulose and is highly specific for this substrate. In the course of this work, the new CBM type was only detected inSpirochaeta, but recently we found sequences with detectable similarity to the module in the draft genomes ofCytophaga fermentansandMahella australiensis, both of which are phylogenetically very distant fromS. thermophilaand noncellulolytic, yet inhabit similar environments. This suggests a possibly broad distribution of the module in nature.

scholarly journals Exposing the Secrets of Two Well-Known Lactobacillus casei Phages, J-1 and PL-1, by Genomic and Structural Analysis

2014 ◽  
Vol 80 (22) ◽  
pp. 7107-7121 ◽  
Author(s):  
Maria Eugenia Dieterle ◽  
Charles Bowman ◽  
Carlos Batthyany ◽  
Esteban Lanzarotti ◽  
Adrián Turjanski ◽  
...  
Keyword(s):  
Lactobacillus Casei ◽  
Sequence Similarity ◽  
Proteolytic Processing ◽  
Mass Spectrometry Analysis ◽  
Carbohydrate Binding ◽  
Content Type ◽  
Related Phage ◽  
Spectrometry Analysis ◽  
C Terminus ◽  
Carbohydrate Binding Modules

ABSTRACTBacteriophage J-1 was isolated in 1965 from an abnormal fermentation of Yakult usingLactobacillus caseistrain Shirota, and a related phage, PL-1, was subsequently recovered from a strain resistant to J-1. Complete genome sequencing shows that J-1 and PL-1 are almost identical, but PL-1 has a deletion of 1.9 kbp relative to J-1, resulting in the loss of four predicted gene products involved in immunity regulation. The structural proteins were identified by mass spectrometry analysis. Similarly to phage A2, two capsid proteins are generated by a translational frameshift and undergo proteolytic processing. The structure of gene product 16 (gp16), a putative tail protein, was modeled based on the crystal structure of baseplate distal tail proteins (Dit) that form the baseplate hub in otherSiphoviridae. However, two regions of the C terminus of gp16 could not be modeled using this template. The first region accounts for the differences between J-1 and PL-1 gp16 and showed sequence similarity to carbohydrate-binding modules (CBMs). J-1 and PL-1 GFP-gp16 fusions bind specifically toLactobacillus casei/paracaseicells, and the addition ofl-rhamnose inhibits binding. J-1 gp16 exhibited a higher affinity than PL-1 gp16 for cell walls ofL. caseiATCC 27139 in phage adsorption inhibition assays, in agreement with differential adsorption kinetics observed for both phages in this strain. The data presented here provide insights into howLactobacillusphages interact with their hosts at the first steps of infection.

scholarly journals Molecular and Biochemical Analyses of the GH44 Module of CbMan5B/Cel44A, a Bifunctional Enzyme from the Hyperthermophilic Bacterium Caldicellulosiruptor bescii

2012 ◽  
Vol 78 (19) ◽  
pp. 7048-7059 ◽  
Author(s):  
Libin Ye ◽  
Xiaoyun Su ◽  
George E. Schmitz ◽  
Young Hwan Moon ◽  
Jing Zhang ◽  
...  
Keyword(s):  
Specific Activity ◽  
Cellulose Hydrolysis ◽  
Thermophilic Bacterium ◽  
Carbohydrate Binding ◽  
Content Type ◽  
Caldicellulosiruptor Bescii ◽  
C Terminus ◽  
Carbohydrate Binding Modules ◽  
Β Sheets ◽  
Biochemical Analyses

ABSTRACTA large polypeptide encoded in the genome of the thermophilic bacteriumCaldicellulosiruptor besciiwas determined to consist of two glycoside hydrolase (GH) modules separated by two carbohydrate-binding modules (CBMs). Based on the detection of mannanase and endoglucanase activities in the N-terminal GH5 and the C-terminal GH44 module, respectively, the protein was designated CbMan5B/Cel44A. A GH5 module with >99% identity from the same organism was characterized previously (X. Su, R. I. Mackie, and I. K. Cann, Appl. Environ. Microbiol.78:2230-2240, 2012); therefore, attention was focused on CbMan5A/Cel44A-TM2 (or TM2), which harbors the GH44 module and the two CBMs. On cellulosic substrates, TM2 had an optimal temperature and pH of 85°C and 5.0, respectively. Although the amino acid sequence of the GH44 module of TM2 was similar to those of other GH44 modules that hydrolyzed cello-oligosaccharides, cellulose, lichenan, and xyloglucan, it was unique that TM2 also displayed modest activity on mannose-configured substrates and xylan. The TM2 protein also degraded Avicel with higher specific activity than activities reported for its homologs. The GH44 catalytic module is composed of a TIM-like domain and a β-sandwich domain, which consists of one β-sheet at the N terminus and nine β-sheets at the C terminus. Deletion of one or more β-sheets from the β-sandwich domain resulted in insoluble proteins, suggesting that the β-sandwich domain is essential for proper folding of the polypeptide. Combining TM2 with three other endoglucanases fromC. besciiled to modest synergistic activities during degradation of cellulose, and based on our results, we propose a model for cellulose hydrolysis and utilization byC. bescii.

scholarly journals Cell Surface Enzyme Attachment Is Mediated by Family 37 Carbohydrate-Binding Modules, Unique to Ruminococcus albus

2008 ◽  
Vol 190 (24) ◽  
pp. 8220-8222 ◽  
Author(s):  
Anat Ezer ◽  
Erez Matalon ◽  
Sadanari Jindou ◽  
Ilya Borovok ◽  
Nof Atamna ◽  
...  
Keyword(s):  
Cell Surface ◽  
Bacterial Cell ◽  
Cellulose Degradation ◽  
Glycoside Hydrolases ◽  
Rumen Bacterium ◽  
Carbohydrate Binding ◽  
Ruminococcus Albus ◽  
System A ◽  
C Terminus ◽  
Carbohydrate Binding Modules

ABSTRACT The rumen bacterium Ruminococcus albus binds to and degrades crystalline cellulosic substrates via a unique cellulose degradation system. A unique family of carbohydrate-binding modules (CBM37), located at the C terminus of different glycoside hydrolases, appears to be responsible both for anchoring these enzymes to the bacterial cell surface and for substrate binding.

scholarly journals Degradation of Granular Starch by the Bacterium Microbacterium aurum Strain B8.A Involves a Modular α-Amylase Enzyme System with FNIII and CBM25 Domains

2015 ◽  
Vol 81 (19) ◽  
pp. 6610-6620 ◽  
Author(s):  
Vincent Valk ◽  
Wieger Eeuwema ◽  
Fean D. Sarian ◽  
Rachel M. van der Kaaij ◽  
Lubbert Dijkhuizen
Keyword(s):  
Enzyme System ◽  
Recombinant Expression ◽  
Potato Starch ◽  
Carbohydrate Binding ◽  
Starch Granules ◽  
Catalytic Core ◽  
Content Type ◽  
C Terminus ◽  
Carbohydrate Binding Modules

ABSTRACTThe bacteriumMicrobacterium aurumstrain B8.A, originally isolated from a potato plant wastewater facility, is able to degrade different types of starch granules. Here we report the characterization of an unusually large, multidomainM. aurumB8.A α-amylase enzyme (MaAmyA). MaAmyA is a 1,417-amino-acid (aa) protein with a predicted molecular mass of 148 kDa. Sequence analysis of MaAmyA showed that its catalytic core is a family GH13_32 α-amylase with the typical ABC domain structure, followed by a fibronectin (FNIII) domain, two carbohydrate binding modules (CBM25), and another three FNIII domains. Recombinant expression and purification yielded an enzyme with the ability to degrade wheat and potato starch granules by introducing pores. Characterization of various truncated mutants of MaAmyA revealed a direct relationship between the presence of CBM25 domains and the ability of MaAmyA to form pores in starch granules, while the FNIII domains most likely function as stable linkers. At the C terminus, MaAmyA carries a 300-aa domain which is uniquely associated with large multidomain amylases; its function remains to be elucidated. We concluded thatM. aurumB8.A employs a multidomain enzyme system to initiate degradation of starch granules via pore formation.

scholarly journals Spatial Distribution and Diverse Metabolic Functions of Lignocellulose-Degrading Uncultured Bacteria as Revealed by Genome-Centric Metagenomics

2018 ◽  
Vol 84 (18) ◽  
Author(s):  
Panagiotis G. Kougias ◽  
Stefano Campanaro ◽  
Laura Treu ◽  
Panagiotis Tsapekos ◽  
Andrea Armani ◽  
...  
Keyword(s):  
Spatial Distribution ◽  
Plant Biomass ◽  
Cellulose Degradation ◽  
Lignocellulose Degradation ◽  
Pig Manure ◽  
Carbohydrate Binding ◽  
Content Type ◽  
Polysaccharide Degradation ◽  
Carbohydrate Binding Modules ◽  
Metabolic Strategies

ABSTRACTThe mechanisms by which specific anaerobic microorganisms remain firmly attached to lignocellulosic material, allowing them to efficiently decompose organic matter, have yet to be elucidated. To circumvent this issue, microbiomes collected from anaerobic digesters treating pig manure and meadow grass were fractionated to separate the planktonic microbes from those adhered to lignocellulosic substrate. Assembly of shotgun reads, followed by a binning process, recovered 151 population genomes, 80 out of which were completely new and were not previously deposited in any database. Genome coverage allowed the identification of microbial spatial distribution in the engineered ecosystem. Moreover, a composite bioinformatic analysis using multiple databases for functional annotation revealed that uncultured members of theBacteroidetesandFirmicutesfollow diverse metabolic strategies for polysaccharide degradation. The structure of cellulosome inFirmicutesspecies can differ depending on the number and functional roles of carbohydrate-binding modules. In contrast, members of theBacteroidetesare able to adhere to and degrade lignocellulose due to the presence of multiple carbohydrate-binding family 6 modules in beta-xylosidase and endoglucanase proteins or S-layer homology modules in unknown proteins. This study combines the concept of variability in spatial distribution with genome-centric metagenomics, allowing a functional and taxonomical exploration of the biogas microbiome.IMPORTANCEThis work contributes new knowledge about lignocellulose degradation in engineered ecosystems. Specifically, the combination of the spatial distribution of uncultured microbes with genome-centric metagenomics provides novel insights into the metabolic properties of planktonic and firmly attached to plant biomass bacteria. Moreover, the knowledge obtained in this study enabled us to understand the diverse metabolic strategies for polysaccharide degradation in different species ofBacteroidetesandClostridiales. Even though structural elements of cellulosome were restricted toClostridialesspecies, our study identified a putative mechanism inBacteroidetesspecies for biomass decomposition, which is based on a gene cluster responsible for cellulose degradation, disaccharide cleavage to glucose, and transport to cytoplasm.

scholarly journals Two Unrelated 8-Vinyl Reductases Ensure Production of Mature Chlorophylls in Acaryochloris marina

2016 ◽  
Vol 198 (9) ◽  
pp. 1393-1400 ◽  
Author(s):  
Guangyu E. Chen ◽  
Andrew Hitchcock ◽  
Philip J. Jackson ◽  
Roy R. Chaudhuri ◽  
Mark J. Dickman ◽  
...  
Keyword(s):  
Transcriptional Control ◽  
Sequence Similarity ◽  
Open Reading Frames ◽  
High Sequence Similarity ◽  
Content Type ◽  
Ethyl Group ◽  
Acaryochloris Marina ◽  
Vinyl Group ◽  
Vinyl Reductase ◽  
Reading Frames

ABSTRACTThe major photopigment of the cyanobacteriumAcaryochloris marinais chlorophylld, while its direct biosynthetic precursor, chlorophylla, is also present in the cell. These pigments, along with the majority of chlorophylls utilized by oxygenic phototrophs, carry an ethyl group at the C-8 position of the molecule, having undergone reduction of a vinyl group during biosynthesis. Two unrelated classes of 8-vinyl reductase involved in the biosynthesis of chlorophylls are known to exist, BciA and BciB. The genome ofAcaryochloris marinacontains open reading frames (ORFs) encoding proteins displaying high sequence similarity to BciA or BciB, although they are annotated as genes involved in transcriptional control (nmrA) and methanogenesis (frhB), respectively. These genes were introduced into an 8-vinyl chlorophylla-producing ΔbciBstrain ofSynechocystissp. strain PCC 6803, and both were shown to restore synthesis of the pigment with an ethyl group at C-8, demonstrating their activities as 8-vinyl reductases. We propose thatnmrAandfrhBbe reassigned asbciAandbciB, respectively; transcript and proteomic analysis ofAcaryochloris marinareveal that bothbciAandbciBare expressed and their encoded proteins are present in the cell, possibly in order to ensure that all synthesized chlorophyll pigment carries an ethyl group at C-8. Potential reasons for the presence of two 8-vinyl reductases in this strain, which is unique for cyanobacteria, are discussed.IMPORTANCEThe cyanobacteriumAcaryochloris marinais the best-studied phototrophic organism that uses chlorophylldfor photosynthesis. Unique among cyanobacteria sequenced to date, its genome contains ORFs encoding two unrelated enzymes that catalyze the reduction of the C-8 vinyl group of a precursor molecule to an ethyl group. Carrying a reduced C-8 group may be of particular importance to organisms containing chlorophylld. Plant genomes also contain orthologs of both of these genes; thus, the bacterial progenitor of the chloroplast may also have contained bothbciAandbciB.

scholarly journals Genome Sequence of the Polysaccharide-Degrading, Thermophilic Anaerobe Spirochaeta thermophila DSM 6192

2010 ◽  
Vol 192 (24) ◽  
pp. 6492-6493 ◽  
Author(s):  
Angel Angelov ◽  
Susanne Liebl ◽  
Meike Ballschmiter ◽  
Mechthild Bömeke ◽  
Rüdiger Lehmann ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Glycoside Hydrolase ◽  
Enzyme System ◽  
Cellulose Degradation ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Free Living ◽  
Genome Data ◽  
Genes Encoding

ABSTRACT Spirochaeta thermophila is a thermophilic, free-living anaerobe that is able to degrade various α- and β-linked sugar polymers, including cellulose. We report here the complete genome sequence of S. thermophila DSM 6192, which is the first genome sequence of a thermophilic, free-living member of the Spirochaetes phylum. The genome data reveal a high density of genes encoding enzymes from more than 30 glycoside hydrolase families, a noncellulosomal enzyme system for (hemi)cellulose degradation, and indicate the presence of a novel carbohydrate-binding module.

scholarly journals Establishment of anIn Vitrod-Cycloserine-Synthesizing System by UsingO-Ureido-l-Serine Synthase and d-Cycloserine Synthetase Found in the Biosynthetic Pathway

2013 ◽  
Vol 57 (6) ◽  
pp. 2603-2612 ◽  
Author(s):  
Narutoshi Uda ◽  
Yasuyuki Matoba ◽  
Takanori Kumagai ◽  
Kosuke Oda ◽  
Masafumi Noda ◽  
...  
Keyword(s):  
Gene Cluster ◽  
High Performance ◽  
Sequence Similarity ◽  
Open Reading Frames ◽  
Homocysteine Thiolactone ◽  
Putative Gene ◽  
Content Type ◽  
Dna Fragment ◽  
Layer Chromatography

ABSTRACTWe have recently cloned a DNA fragment containing a gene cluster that is responsible for the biosynthesis of an antituberculosis antibiotic,d-cycloserine. The gene cluster is composed of 10 open reading frames, designateddcsAtodcsJ. Judging from the sequence similarity between each putative gene product and known proteins, DcsC, which displays high homology to diaminopimelate epimerase, may catalyze the racemization ofO-ureidoserine. DcsD is similar toO-acetylserine sulfhydrylase, which generatesl-cysteine usingO-acetyl-l-serine with sulfide, and therefore, DcsD may be a synthase to generateO-ureido-l-serine usingO-acetyl-l-serine and hydroxyurea. DcsG, which exhibits similarity to a family of enzymes with an ATP-grasp fold, may be an ATP-dependent synthetase convertingO-ureido-d-serine intod-cycloserine. In the present study, to characterize the enzymatic functions of DcsC, DcsD, and DcsG, each protein was overexpressed inEscherichia coliand purified to near homogeneity. The biochemical function of each of the reactions catalyzed by these three proteins was verified by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and, in some cases, mass spectrometry. The results from this study demonstrate that by using a mixture of the three purified enzymes and the two commercially available substratesO-acetyl-l-serine and hydroxyurea, synthesis ofd-cycloserine was successfully attained. Thesein vitrostudies yield the conclusion that DcsD and DcsG are necessary for the syntheses ofO-ureido-l-serine andd-cycloserine, respectively. DcsD was also able to catalyze the synthesis ofl-cysteine when sulfide was added instead of hydroxyurea. Furthermore, the present study shows that DcsG can also form other cyclicd-amino acid analogs, such asd-homocysteine thiolactone.

scholarly journals Functional Screening of a Metagenomic Library Reveals Operons Responsible for Enhanced Intestinal Colonization by Gut Commensal Microbes

2013 ◽  
Vol 79 (12) ◽  
pp. 3829-3838 ◽  
Author(s):  
Mi Young Yoon ◽  
Kang-Mu Lee ◽  
Yujin Yoon ◽  
Junhyeok Go ◽  
Yongjin Park ◽  
...  
Keyword(s):  
Bac Library ◽  
Metagenomic Library ◽  
Artificial Chromosome ◽  
Open Reading Frames ◽  
Functional Screening ◽  
Sequence Alignments ◽  
Protein Coding ◽  
Content Type ◽  
Usage Analysis ◽  
Functional Screens

ABSTRACTEvidence suggests that gut microbes colonize the mammalian intestine through propagation as an adhesive microbial community. A bacterial artificial chromosome (BAC) library of murine bowel microbiota DNA in the surrogate hostEscherichia coliDH10B was screened for enhanced adherence capability. Two out of 5,472 DH10B clones, 10G6 and 25G1, exhibited enhanced capabilities to adhere to inanimate surfaces in functional screens. DNA segments inserted into the 10G6 and 25G1 clones were 52 and 41 kb and included 47 and 41 protein-coding open reading frames (ORFs), respectively. DNA sequence alignments, tetranucleotide frequency, and codon usage analysis strongly suggest that these two DNA fragments are derived from species belonging to the genusBacteroides. Consistent with this finding, a large portion of the predicted gene products were highly homologous to those ofBacteroidesspp. Transposon mutagenesis and subsequent experiments that involved heterologous expression identified two operons associated with enhanced adherence.E. colistrains transformed with the 10a or 25b operon adhered to the surface of intestinal epithelium and colonized the mouse intestine more vigorously than did the control strain. This study has revealed the genetic determinants of unknown commensals (probably resemblingBacteroidesspecies) that enhance the ability of the bacteria to colonize the murine bowel.

scholarly journals Complete Genome Sequence of Streptococcus pneumoniae Strain Rx1, a Hex Mismatch Repair-Deficient Standard Transformation Recipient

2021 ◽  
Vol 10 (41) ◽  
Author(s):  
Anna Maria Cuppone ◽  
Lorenzo Colombini ◽  
Valeria Fox ◽  
David Pinzauti ◽  
Francesco Santoro ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Mismatch Repair ◽  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Gc Content ◽  
Open Reading Frames ◽  
Circular Chromosome ◽  
Content Type ◽  
Sequencing Technologies

The complete genome sequence of Streptococcus pneumoniae strain Rx1, a Hex mismatch repair-deficient standard transformation recipient, was obtained by combining Nanopore and Illumina sequencing technologies. The genome consists of a 2.03-Mb circular chromosome, with 2,054 open reading frames and a GC content of 39.72%.

Sign in / Sign up

Export Citation Format

Share Document