scholarly journals Two Alternative Pathways for the Synthesis of the Rare Compatible Solute Mannosylglucosylglycerate in Petrotoga mobilis

2010 ◽  
Vol 192 (6) ◽  
pp. 1624-1633 ◽  
Author(s):  
Chantal Fernandes ◽  
Vitor Mendes ◽  
Joana Costa ◽  
Nuno Empadinhas ◽  
Carla Jorge ◽  
...  
Keyword(s):  
Sequence Homology ◽  
Thermotoga Maritima ◽  
Functional Characterization ◽  
Compatible Solute ◽  
Homologous Gene ◽  
Gene Encoding ◽  
Alternative Pathways ◽  
Cell Extracts ◽  
Genes Encoding

ABSTRACT The compatible solute mannosylglucosylglycerate (MGG), recently identified in Petrotoga miotherma, also accumulates in Petrotoga mobilis in response to hyperosmotic conditions and supraoptimal growth temperatures. Two functionally connected genes encoding a glucosyl-3-phosphoglycerate synthase (GpgS) and an unknown glycosyltransferase (gene Pmob_1143), which we functionally characterized as a mannosylglucosyl-3-phosphoglycerate synthase and designated MggA, were identified in the genome of Ptg. mobilis. This enzyme used the product of GpgS, glucosyl-3-phosphoglycerate (GPG), as well as GDP-mannose to produce mannosylglucosyl-3-phosphoglycerate (MGPG), the phosphorylated precursor of MGG. The MGPG dephosphorylation was determined in cell extracts, and the native enzyme was partially purified and characterized. Surprisingly, a gene encoding a putative glucosylglycerate synthase (Ggs) was also identified in the genome of Ptg. mobilis, and an active Ggs capable of producing glucosylglycerate (GG) from ADP-glucose and d-glycerate was detected in cell extracts and the recombinant enzyme was characterized, as well. Since GG has never been identified in this organism nor was it a substrate for the MggA, we anticipated the existence of a nonphosphorylating pathway for MGG synthesis. We putatively identified the corresponding gene, whose product had some sequence homology with MggA, but it was not possible to recombinantly express a functional enzyme from Ptg. mobilis, which we named mannosylglucosylglycerate synthase (MggS). In turn, a homologous gene from Thermotoga maritima was successfully expressed, and the synthesis of MGG was confirmed from GDP-mannose and GG. Based on the measurements of the relevant enzyme activities in cell extracts and on the functional characterization of the key enzymes, we propose two alternative pathways for the synthesis of the rare compatible solute MGG in Ptg. mobilis.

scholarly journals Identification and Functional Characterization of the Gene Cluster Responsible for Fusaproliferin Biosynthesis in Fusarium proliferatum

Toxins ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 468
Author(s):  
Asja Ćeranić ◽  
Thomas Svoboda ◽  
Franz Berthiller ◽  
Michael Sulyok ◽  
Jonathan Matthew Samson ◽  
...  
Keyword(s):  
Fusarium Species ◽  
Functional Characterization ◽  
Fusarium Proliferatum ◽  
Cytochrome P450s ◽  
Gene Encoding ◽  
Knock Out ◽  
Genes Encoding ◽  
Terpenoid Synthase ◽  
Taxonomic Groups

The emerging mycotoxin fusaproliferin is produced by Fusarium proliferatum and other related Fusarium species. Several fungi from other taxonomic groups were also reported to produce fusaproliferin or the deacetylated derivative, known as siccanol or terpestacin. Here, we describe the identification and functional characterization of the Fusarium proliferatum genes encoding the fusaproliferin biosynthetic enzymes: a terpenoid synthase, two cytochrome P450s, a FAD-oxidase and an acetyltransferase. With the exception of one gene encoding a CYP450 (FUP2, FPRN_05484), knock-out mutants of the candidate genes could be generated, and the production of fusaproliferin and intermediates was tested by LC-MS/MS. Inactivation of the FUP1 (FPRN_05485) terpenoid synthase gene led to complete loss of fusaproliferin production. Disruption of a putative FAD-oxidase (FUP4, FPRN_05486) did not only affect oxidation of preterpestacin III to terpestacin, but also of new side products (11-oxo-preterpstacin and terpestacin aldehyde). In the knock-out strains lacking the predicted acetyltransferase (FUP5, FPRN_05487) fusaproliferin was no longer formed, but terpestacin was found at elevated levels. A model for the biosynthesis of fusaproliferin and of novel derivatives found in mutants is presented.

scholarly journals Characterization of the Genes Encoding d-Amino Acid Transaminase and Glutamate Racemase, Two d-Glutamate Biosynthetic Enzymes of Bacillus sphaericus ATCC 10208

1998 ◽  
Vol 180 (16) ◽  
pp. 4319-4323 ◽  
Author(s):  
Ian G. Fotheringham ◽  
Stefan A. Bledig ◽  
Paul P. Taylor
Keyword(s):  
Cell Wall ◽  
Amino Acid ◽  
Sequence Homology ◽  
Bacillus Sphaericus ◽  
Primary Sequence ◽  
Gene Encoding ◽  
Sequence Identity ◽  
Glutamate Racemase ◽  
Genes Encoding

ABSTRACT In Bacillus sphaericus and other Bacillusspp., d-amino acid transaminase has been considered solely responsible for biosynthesis of d-glutamate, an essential component of cell wall peptidoglycan, in contrast to the glutamate racemase employed by many other bacteria. We report here the cloning of the dat gene encoding d-amino acid transaminase and the glr gene encoding a glutamate racemase from B. sphaericus ATCC 10208. The glrgene encodes a 28.8-kDa protein with 40 to 50% sequence identity to the glutamate racemases of Lactobacillus,Pediococcus, and Staphylococcus species. Thedat gene encodes a 31.4-kDa peptide with 67% primary sequence homology to the d-amino acid transaminase of the thermophilic Bacillus sp. strain YM1.

Identification and functional characterization of levS, a gene encoding for a levansucrase from Leuconostoc mesenteroides NRRL B-512 F

Gene ◽  
2006 ◽  
Vol 376 (1) ◽  
pp. 59-67 ◽  
Author(s):  
Sandra Morales-Arrieta ◽  
Maria Elena Rodríguez ◽  
Lorenzo Segovia ◽  
Agustín López-Munguía ◽  
Clarita Olvera-Carranza
Keyword(s):  
Leuconostoc Mesenteroides ◽  
Functional Characterization ◽  
Gene Encoding

scholarly journals Cloning and functional characterization of mammalian homologues of the COPII component Sec23.

1996 ◽  
Vol 7 (10) ◽  
pp. 1535-1546 ◽  
Author(s):  
J P Paccaud ◽  
W Reith ◽  
J L Carpentier ◽  
M Ravazzola ◽  
M Amherdt ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Cdna Clones ◽  
Restrictive Temperature ◽  
Rnase Protection ◽  
Cell Extracts ◽  
Exact Function ◽  
Mammalian Protein

We screened a human cDNA library with a probe derived from a partial SEC23 mouse homologue and isolated two different cDNA clones (hSec23A and hSec23B) encoding proteins of a predicted molecular mass of 85 kDa. hSec23Ap and hSec23Bp were 85% identical and shared 48% identity with the yeast Sec23p. Affinity-purified anti-hSec23A recognized a protein of approximately 85 kDa on immunoblots of human, mouse, and rat cell extracts but did not recognize yeast Sec23p. Cytosolic hSec23Ap migrated with an apparent molecular weight of 350 kDa on a gel filtration column, suggesting that it is part of a protein complex. By immunoelectron microscopy, hSec23Ap was found essentially in the ribosome-free transitional face of the endoplasmic reticulum (ER) and associated vesicles. hSec23Ap is a functional homologue of the yeast Sec23p as the hSec23A isoform complemented the temperature sensitivity of the Saccharomyces cerevisiae sec23-1 mutation at a restrictive temperature of 34 degrees C. RNase protection assays indicated that both hSec23 isoforms are coexpressed in various human tissues, although at a variable ratio. Our data demonstrate that hSec23Ap is the functional human counterpart of the yeast COPII component Sec23p and suggest that it plays a similar role in mammalian protein export from the ER. The exact function of hSec23Bp remains to be determined.

scholarly journals Identification of Streptococci to Species Level by Sequencing the Gene Encoding the Manganese-Dependent Superoxide Dismutase

1998 ◽  
Vol 36 (1) ◽  
pp. 41-47 ◽  
Author(s):  
Claire Poyart ◽  
Gilles Quesne ◽  
Stephane Coulon ◽  
Patrick Berche ◽  
Patrick Trieu-Cuot
Keyword(s):  
Superoxide Dismutase ◽  
Pcr Assay ◽  
Degenerate Primers ◽  
Gene Encoding ◽  
Genes Encoding ◽  
Type Strains ◽  
Internal Fragment ◽  
Rrna Sequences ◽  
16S Rrna Sequences

We have used a PCR assay based on the use of degenerate primers in order to characterize an internal fragment (sodAint ) representing approximately 85% of the genes encoding the manganese-dependent superoxide dismutase in various streptococcal type strains (S. acidominimus,S. agalactiae, S. alactolyticus, S. anginosus, S. bovis, S. constellatus,S. canis, S. cricetus, S. downei,S. dysgalactiae, S. equi subsp.equi, S. equi subsp. zooepidemicus,S. equinus, S. gordonii, S. iniae,S. intermedius, S. mitis, S. mutans, S. oralis, S. parasanguis,S. pneumoniae, S. porcinus, S. pyogenes, S. salivarius, S. sanguis,S. sobrinus, S. suis, S. thermophilus, and S. vestibularis). Phylogenetic analysis of these sodAint fragments yields an evolutionary tree having a topology similar to that of the tree constructed with the 16S rRNA sequences. We have shown that clinical isolates could be identified by determining the positions of theirsodAint fragments on the phylogenetic tree of the sodAint fragments of the type species. We propose this method for the characterization of strains that cannot be assigned to a species on the basis of their conventional phenotypic reactions.

Cloning and functional characterization of a superfamily of microbial inwardly rectifying potassium channels

2006 ◽  
Vol 26 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Si Sun ◽  
Jo Han Gan ◽  
Jennifer J. Paynter ◽  
Stephen J. Tucker
Keyword(s):  
Functional Properties ◽  
Functional Characterization ◽  
Unicellular Eukaryote ◽  
Channel Blockers ◽  
E Coli ◽  
Inwardly Rectifying Potassium Channels ◽  
Genes Encoding ◽  
Functionally Diverse ◽  
Inwardly Rectifying

Our understanding of the mammalian inwardly rectifying family of K+ channels (Kir family) has recently been advanced by X-ray crystal structures of two homologous prokaryotic orthologs (KirBac1.1 and KirBac3.1). However, the functional properties of these KirBac channels are still poorly understood. To address this problem, we cloned and characterized genes encoding KirBac orthologs from a wide variety of different prokaryotes and a simple unicellular eukaryote. The functional properties of these KirBacs were then examined by growth complementation in a K+ uptake-deficient strain of Escherichia coli (TK2420). Whereas some KirBac genes exhibited robust growth complementation, others either did not complement or showed temperature-dependent complementation including KirBac1.1 and KirBac3.1. In some cases, KirBac expression was also toxic to the growth of E. coli. The KirBac family exhibited a range of sensitivity to the K+ channel blockers Ba2+ and Cs+ as well as differences in their ability to grow on very low-K+ media, thus demonstrating major differences in their permeation properties. These results reveal the existence of a functionally diverse superfamily of microbial KirBac genes and present an excellent resource for the structural and functional analysis of this class of K+ channels. Furthermore, the complementation assay used in this study provides a simple and robust method for the functional characterization of a range of prokaryotic K+ channels that are difficult to study by traditional methods.

scholarly journals Cloning and functional characterization of the gene encoding the transcription factor Acel in the basidiomycete Phanerochaete chrysosporium

2006 ◽  
Vol 39 (4) ◽  
Author(s):  
RUBÉN POLANCO ◽  
PAULO CANESSA ◽  
ALEXIS RIVAS ◽  
LUIS F LARRONDO ◽  
SERGIO LOBOS ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Phanerochaete Chrysosporium ◽  
Functional Characterization ◽  
Gene Encoding
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