scholarly journals Investigating the Function of an Arabinan Utilization Locus Isolated from a Termite Gut Community

2014 ◽  
Vol 81 (1) ◽  
pp. 31-39 ◽  
Author(s):  
Grégory Arnal ◽  
Géraldine Bastien ◽  
Nelly Monties ◽  
Anne Abot ◽  
Véronique Anton Leberre ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Plant Biomass ◽  
Bioinformatic Analysis ◽  
Open Reading Frames ◽  
Glycoside Hydrolases ◽  
Functional Screening ◽  
Content Type ◽  
Termite Gut ◽  
Pentose Sugar

ABSTRACTBiocatalysts are essential for the development of bioprocesses efficient for plant biomass degradation. Previously, a metagenomic clone containing DNA from termite gut microbiota was pinpointed in a functional screening that revealed the presence of arabinofuranosidase activity. Subsequent genetic and bioinformatic analysis revealed that the DNA fragment belonged to a member of the genusBacteroidesand encoded 19 open reading frames (ORFs), and annotation suggested the presence of hypothetical transporter and regulator proteins and others involved in the catabolism of pentose sugar. In this respect and considering the phenotype of the metagenomic clone, it was noted that among the ORFs, there are four putative arabinose-specific glycoside hydrolases, two from family GH43 and two from GH51. In this study, a thorough bioinformatics analysis of the metagenomic clone gene cluster has been performed and the four aforementioned glycoside hydrolases have been characterized. Together, the results provide evidence that the gene cluster is a polysaccharide utilization locus dedicated to the breakdown of the arabinan component in pectin and related substrates. Characterization of the two GH43 and the two GH51 glycoside hydrolases has revealed that each of these enzymes displays specific catalytic capabilities and that when these are combined the enzymes act synergistically, increasing the efficiency of arabinan degradation.

scholarly journals Identification and Characterization of a Halotolerant, Cold-Active Marine Endo-β-1,4-Glucanase by Using Functional Metagenomics of Seaweed-Associated Microbiota

2014 ◽  
Vol 80 (16) ◽  
pp. 4958-4967 ◽  
Author(s):  
Marjolaine Martin ◽  
Sophie Biver ◽  
Sébastien Steels ◽  
Tristan Barbeyron ◽  
Murielle Jam ◽  
...  
Keyword(s):  
Metagenomic Library ◽  
Functional Screening ◽  
Sample Collection ◽  
Prolonged Incubation ◽  
Content Type ◽  
Cold Active ◽  
Esterase Loci ◽  
Collection Period ◽  
Identification And Characterization

ABSTRACTA metagenomic library was constructed from microorganisms associated with the brown algaAscophyllum nodosum. Functional screening of this library revealed 13 novel putative esterase loci and two glycoside hydrolase loci. Sequence and gene cluster analysis showed the wide diversity of the identified enzymes and gave an idea of the microbial populations present during the sample collection period. Lastly, an endo-β-1,4-glucanase having less than 50% identity to sequences of known cellulases was purified and partially characterized, showing activity at low temperature and after prolonged incubation in concentrated salt solutions.

scholarly journals Characterization of Two Virulent Phages of Lactobacillus plantarum

2012 ◽  
Vol 78 (24) ◽  
pp. 8719-8734 ◽  
Author(s):  
Mariángeles Briggiler Marcó ◽  
Josiane E. Garneau ◽  
Denise Tremblay ◽  
Andrea Quiberoni ◽  
Sylvain Moineau
Keyword(s):  
Lactobacillus Plantarum ◽  
Corn Silage ◽  
Open Reading Frames ◽  
High Identity ◽  
Content Type ◽  
Defense Systems ◽  
Double Stranded Dna ◽  
Type Phage ◽  
Reading Frames

ABSTRACTWe characterized twoLactobacillus plantarumvirulent siphophages, ATCC 8014-B1 (B1) and ATCC 8014-B2 (B2), previously isolated from corn silage and anaerobic sewage sludge, respectively. Phage B2 infected two of the eightL. plantarumstrains tested, while phage B1 infected three. Phage adsorption was highly variable depending on the strain used. Phage defense systems were found in at least twoL. plantarumstrains, LMG9211 and WCSF1. The linear double-stranded DNA genome of thepac-type phage B1 had 38,002 bp, a G+C content of 47.6%, and 60 open reading frames (ORFs). Surprisingly, the phage B1 genome has 97% identity with that ofPediococcus damnosusphage clP1 and 77% identity with that ofL. plantarumphage JL-1; these phages were isolated from sewage and cucumber fermentation, respectively. The double-stranded DNA (dsDNA) genome of thecos-type phage B2 had 80,618 bp, a G+C content of 36.9%, and 127 ORFs with similarities to those ofBacillusandLactobacillusstrains as well as phages. Some phage B2 genes were similar to ORFs fromL. plantarumphage LP65 of theMyoviridaefamily. Additionally, 6 tRNAs were found in the phage B2 genome. Protein analysis revealed 13 (phage B1) and 9 (phage B2) structural proteins. To our knowledge, this is the first report describing such high identity between phage genomes infecting different genera of lactic acid bacteria.

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 Characterization of the Enterobacter Phage vB_EclM_CIP9

2020 ◽  
Vol 9 (13) ◽  
Author(s):  
Klara Wang ◽  
Marielou G. Tamayo ◽  
Tiffany V. Penner ◽  
Bradley W. M. Cook ◽  
Deborah A. Court ◽  
...  
Keyword(s):  
Enterobacter Cloacae ◽  
Opportunistic Pathogen ◽  
Multidrug Resistant ◽  
Open Reading Frames ◽  
Resistant Isolate ◽  
Content Type ◽  
Hospital Acquired Infections ◽  
Hospital Acquired ◽  
Reading Frames

Enterobacter cloacae is an opportunistic pathogen that causes hospital-acquired infections in immunocompromised patients. Here, we describe vB_EclM_CIP9, a novel Enterobacter phage that infects a multidrug-resistant isolate of E. cloacae. Phage vB_EclM_CIP9 is a myovirus that has a 174,924-bp genome, with 296 predicted open reading frames.

scholarly journals Construction and Application of a Functional Library of Cytochrome P450 Monooxygenases from the Filamentous Fungus Aspergillus oryzae

2011 ◽  
Vol 77 (9) ◽  
pp. 3147-3150 ◽  
Author(s):  
K. H. M. Nazmul Hussain Nazir ◽  
Hirofumi Ichinose ◽  
Hiroyuki Wariishi
Keyword(s):  
Cytochrome P450 ◽  
Aspergillus Oryzae ◽  
Cytochrome P450 Monooxygenases ◽  
Functional Screening ◽  
Content Type ◽  
P450 Monooxygenases ◽  
Rapid Characterization ◽  
Catalytic Functions ◽  
First Time

ABSTRACTA functional library of cytochrome P450 monooxygenases fromAspergillus oryzae(AoCYPs) was constructed in which 121 isoforms were coexpressed with yeast NADPH-cytochrome P450 oxidoreductase inSaccharomyces cerevisiae. Using this functional library, novel catalytic functions of AoCYPs, such as catalytic potentials of CYP57B3 against genistein, were elucidated for the first time. Comprehensive functional screening promises rapid characterization of catalytic potentials and utility of AoCYPs.

scholarly journals wksl3, a New Biocontrol Agent for Salmonella enterica Serovars Enteritidis and Typhimurium in Foods: Characterization, Application, Sequence Analysis, and Oral Acute Toxicity Study

2013 ◽  
Vol 79 (6) ◽  
pp. 1956-1968 ◽  
Author(s):  
Hyun-Wol Kang ◽  
Jae-Won Kim ◽  
Tae-Sung Jung ◽  
Gun-Jo Woo
Keyword(s):  
Sequence Analysis ◽  
Salmonella Enterica ◽  
Clinical Symptoms ◽  
Bioinformatic Analysis ◽  
Open Reading Frames ◽  
Oral Toxicity ◽  
Content Type ◽  
Log Reduction ◽  
Food Borne ◽  
The Republic

ABSTRACTOf theSalmonella entericaserovars,S. Enteritidis andS. Typhimurium are responsible for most of theSalmonellaoutbreaks implicated in the consumption of contaminated foods in the Republic of Korea. Because of the widespread occurrence of antimicrobial-resistantSalmonellain foods and food processing environments, bacteriophages have recently surfaced as an alternative biocontrol tool. In this study, we isolated a virulent bacteriophage (wksl3) that could specifically infectS. Enteritidis,S. Typhimurium, and several additional serovars. Transmission electron microscopy revealed that phage wksl3 belongs to the familySiphoviridae. Complete genome sequence analysis and bioinformatic analysis revealed that the DNA of phage wksl3 is composed of 42,766 bp with 64 open reading frames. Since it does not encode any phage lysogeny factors, toxins, pathogen-related genes, or food-borne allergens, phage wksl3 may be considered a virulent phage with no side effects. Analysis of genetic similarities between phage wksl3 and four of its relatives (SS3e, vB_SenS-Ent1, SE2, and SETP3) allowed wksl3 to be categorized as a SETP3-like phage. A single-dose test of oral toxicity with BALB/c mice resulted in no abnormal clinical observations. Moreover, phage application to chicken skin at 8°C resulted in an about 2.5-log reduction in the number ofSalmonellabacteria during the test period. The strong, stable lytic activity, the significant reduction of the number ofS. Enteritidis bacteria after application to food, and the lack of clinical symptoms of this phage suggest that wksl3 may be a useful agent for the protection of foods againstS. Enteritidis andS. Typhimurium contamination.

scholarly journals High-Throughput Recovery and Characterization of Metagenome-Derived Glycoside Hydrolase-Containing Clones as a Resource for Biocatalyst Development

mSystems ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Zachary Armstrong ◽  
Feng Liu ◽  
Sam Kheirandish ◽  
Hong-Ming Chen ◽  
Keith Mewis ◽  
...  
Keyword(s):  
High Throughput ◽  
Glycoside Hydrolase ◽  
Plant Biomass ◽  
Functional Characterization ◽  
Environmental Dna ◽  
Functional Screening ◽  
Biomass Degradation ◽  
Genomic Context ◽  
Or Gene

ABSTRACT Functional metagenomics is a powerful tool for both the discovery and development of biocatalysts. This study presents the high-throughput functional screening of 22 large-insert fosmid libraries containing over 300,000 clones sourced from natural and engineered ecosystems, characterization of active clones, and a demonstration of the utility of recovered genes or gene cassettes in the development of novel biocatalysts. Screening was performed in a 384-well-plate format with the fluorogenic substrate 4-methylumbelliferyl cellobioside, which releases a fluorescent molecule when cleaved by β-glucosidases or cellulases. The resulting set of 164 active clones was subsequently interrogated for substrate preference, reaction mechanism, thermal stability, and optimal pH. The environmental DNA harbored within each active clone was sequenced, and functional annotation revealed a cornucopia of carbohydrate-degrading enzymes. Evaluation of genomic-context information revealed both synteny and polymer-targeting loci within a number of sequenced clones. The utility of these fosmids was then demonstrated by identifying clones encoding activity on an unnatural glycoside (4-methylumbelliferyl 6-azido-6-deoxy-β-d-galactoside) and transforming one of the identified enzymes into a glycosynthase capable of forming taggable disaccharides. IMPORTANCE The generation of new biocatalysts for plant biomass degradation and glycan synthesis has typically relied on the characterization and investigation of one or a few enzymes at a time. By coupling functional metagenomic screening and high-throughput functional characterization, we can progress beyond the current scale of catalyst discovery and provide rapid annotation of catalyst function. By functionally screening environmental DNA from many diverse sources, we have generated a suite of active glycoside hydrolase-containing clones and demonstrated their reaction parameters. We then demonstrated the utility of this collection through the generation of a new catalyst for the formation of azido-modified glycans. Further interrogation of this collection of clones will expand our biocatalytic toolbox, with potential application to biomass deconstruction and synthesis of glycans.

scholarly journals Genetic Characterization of Plasmid-Borne blaOXA-58 in Distinct Acinetobacter Species

mSphere ◽  
2019 ◽  
Vol 4 (5) ◽  
Author(s):  
Adriana P. Matos ◽  
Rodrigo Cayô ◽  
Luiz G. P. Almeida ◽  
Ana Paula Streling ◽  
Carolina S. Nodari ◽  
...  
Keyword(s):  
Open Reading Frames ◽  
South American ◽  
Acinetobacter Species ◽  
Content Type ◽  
Clinical Strains ◽  
Carbapenem Resistant ◽  
Encoding Genes ◽  
Reading Frames ◽  
Over Time

ABSTRACT We characterize by whole-plasmid-sequence (WPS) two-plasmid-borne blaOXA-58 obtained from Acinetobacter seifertii (Asp-1069) and A. baumannii (Acb-45063) clinical strains recovered 17 years apart from distinct Brazilian regions. Multilocus sequence type (MLST) analysis showed that the Asp-1069 and Acb-45063 strains belong to ST551 and ST15/CC15, respectively. WPS analysis demonstrated that blaOXA-58 was located in two distinct plasmids named pAs1069_a (24,672 bp/44 open reading frames [ORFs]) and pAb45063_b (19,808 bp/24 ORFs), which belong to the GR8/GR23 (repAci23) and GR4 (repAci4) incompatibility groups, respectively. The genetic environments surrounding blaOXA-58 revealed that it was flanked by two intact ISAba3 copies on pAb45063_b, which differed from pAs1069_a. In the latter, the upstream ISAba3 copy was truncated by insertion of ISAba825 element. Although Re27-specific recombination sites were found adjacent to ISAba3-blaOXA-58-ISAba3 arrangement on pAb45063_b, such structures were absent on pAs1069_a. The conserved ISAba125-araC1-lysE arrangement was disrupted by TnaphA6 harboring the aminoglycosides resistance gene aphA6 on pAs1069_a, while an IS26-blaTEM-1-aac(3)-IIa-IS26 genetic structure was found upstream from ISAba3-blaOXA-58-ISAba3 on pAb45063_b. Other two plasmids, pAb45063_a (183,767 bp/209 ORFs) and pAs1069_b (13,129 bp/14 ORFs), were also found in the OXA-58-producing Acinetobacter species strains, harboring the strA and strB genes and the sul2 gene, which confer resistance to streptomycin and sulfonamides, respectively. The plasmid-mediated virulence factors corresponding to genes tonB, spl, glmM, ppa, sulP, and map were found in both strains, as well distinct toxin-antitoxin system-encoding genes stbD and relE (pAs1069_a), brnT and brnA (pAb45063_b), and xreE (pAb45063_a). Although infrequently reported in Brazil, plasmid-borne blaOXA-58 showed a complex and diverse genetic backbone that confers stability in different Acinetobacter species that have been isolated from nosocomial settings over time. IMPORTANCE Although the blaOXA-58 gene has been infrequently described in Brazil, contrasting with other bordering South American countries, we verified the maintenance of this resistance determinant over time among carbapenem-resistant Acinetobacter species isolates, not only in nosocomial settings but also in the environment. In addition, to the best of our knowledge, this is the first study to have used WPS analysis to evaluate the genetic surroundings of blaOXA-58 in Brazil. Moreover, the A. seifertii and A. baumannii clinical strains evaluated in this study were recovered 17 years apart in hospitals located in distinct Brazilian geographic regions.

scholarly journals Involvement of a Natural Fusion of a Cytochrome P450 and a Hydrolase in Mycophenolic Acid Biosynthesis

2012 ◽  
Vol 78 (14) ◽  
pp. 4908-4913 ◽  
Author(s):  
Bjarne Gram Hansen ◽  
Ewelina Mnich ◽  
Kristian Fog Nielsen ◽  
Jakob Blæsbjerg Nielsen ◽  
Morten Thrane Nielsen ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Gene Cluster ◽  
Mycophenolic Acid ◽  
Polyketide Synthase ◽  
Fusion Gene ◽  
Bioinformatic Analysis ◽  
Terminal Region ◽  
Ring Closure ◽  
Content Type ◽  
Penicillium Brevicompactum

ABSTRACTMycophenolic acid (MPA) is a fungal secondary metabolite and the active component in several immunosuppressive pharmaceuticals. The gene cluster coding for the MPA biosynthetic pathway has recently been discovered inPenicillium brevicompactum, demonstrating that the first step is catalyzed by MpaC, a polyketide synthase producing 5-methylorsellinic acid (5-MOA). However, the biochemical role of the enzymes encoded by the remaining genes in the MPA gene cluster is still unknown. Based on bioinformatic analysis of the MPA gene cluster, we hypothesized that the step following 5-MOA production in the pathway is carried out by a natural fusion enzyme MpaDE, consisting of a cytochrome P450 (MpaD) in the N-terminal region and a hydrolase (MpaE) in the C-terminal region. We verified that the fusion gene is indeed expressed inP. brevicompactumby obtaining full-length sequence of thempaDEcDNA prepared from the extracted RNA. Heterologous coexpression ofmpaCand the fusion genempaDEin the MPA-nonproducerAspergillus nidulansresulted in the production of 5,7-dihydroxy-4-methylphthalide (DHMP), the second intermediate in MPA biosynthesis. Analysis of the strain coexpressingmpaCand thempaDpart ofmpaDEshows that the P450 catalyzes hydroxylation of 5-MOA to 4,6-dihydroxy-2-(hydroxymethyl)-3-methylbenzoic acid (DHMB). DHMB is then converted to DHMP, and our results suggest that the hydrolase domain aids this second step by acting as a lactone synthase that catalyzes the ring closure. Overall, the chimeric enzyme MpaDE provides insight into the genetic organization of the MPA biosynthesis pathway.

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