VanE, a New Type of Acquired Glycopeptide Resistance in Enterococcus faecalis BM4405

1999 ◽  
Vol 43 (9) ◽  
pp. 2161-2164 ◽  
Author(s):  
Marguerite Fines ◽  
Bruno Perichon ◽  
Peter Reynolds ◽  
Daniel F. Sahm ◽  
Patrice Courvalin
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Enterococcus Faecalis ◽  
Resistance Genes ◽  
Degenerate Primers ◽  
Glycopeptide Resistance ◽  
Pcr Product ◽  
New Type ◽  
Total Dna ◽  
Low Levels

ABSTRACT Enterococcus faecalis BM4405 was resistant to low levels of vancomycin (MIC, 16 μg/ml) and was susceptible to teicoplanin (MIC, 0.5 μg/ml). No PCR product was obtained when the total DNA of this clinical isolate was used as a template with primers specific for glycopeptide resistance genes vanA,vanB, vanC, and vanD. However, a 604-bp PCR fragment was obtained when V1 and V2 degenerate primers were used and total DNA was digested with HindIII as a template. The product was cloned and sequenced. The deduced amino acid sequence had greater identity (55%) with VanC than with VanA (45%), VanB (43%), or VanD (44%). This was consistent with the fact that BM4405 synthesized peptidoglycan precursors that terminated ind-serine residues. After induction with vancomycin, weakd,d-dipeptidase and penicillin-insensitived,d-carboxypeptidase activities were detected in cytoplasmic extracts of BM4405, whereas a serine racemase activity was found in the membrane preparation. This new type of acquired glycopeptide resistance was named VanE.

scholarly journals VanD-type glycopeptide-resistant Enterococcus faecium BM4339.

1997 ◽  
Vol 41 (9) ◽  
pp. 2016-2018 ◽  
Author(s):  
B Perichon ◽  
P Reynolds ◽  
P Courvalin
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Enterococcus Faecium ◽  
Glycopeptide Resistance ◽  
Resistance Phenotype ◽  
Resistance Proteins ◽  
Genes Encoding ◽  
New Type ◽  
Low Levels

Enterococcus faecium BM4339 was constitutively resistant to vancomycin (MIC, 64 microg/ml) and to low levels of teicoplanin (MIC, 4 microg/ml). A 605-bp product obtained with the V1 and V2 primers for amplification of genes encoding D-Ala:D-Ala ligases and related glycopeptide resistance proteins was sequenced after cloning. The deduced amino acid sequence had 69% identity with VanA and VanB and 43% identity with VanC, consistent with the finding that BM4339 synthesized peptidoglycan precursors terminating in D-lactate. This new type of glycopeptide resistance phenotype was designated VanD.

scholarly journals d-Ala-d-Ser VanN-Type Transferable Vancomycin Resistance in Enterococcus faecium

2011 ◽  
Vol 55 (10) ◽  
pp. 4606-4612 ◽  
Author(s):  
François Lebreton ◽  
Florence Depardieu ◽  
Nancy Bourdon ◽  
Marguerite Fines-Guyon ◽  
Pierre Berger ◽  
...  
Keyword(s):  
Enterococcus Faecium ◽  
Constitutive Expression ◽  
Degenerate Primers ◽  
Glycopeptide Resistance ◽  
Content Type ◽  
Pcr Product ◽  
Low Levels ◽  
Rapid Amplification ◽  
Race Pcr ◽  
Tail Pcr

ABSTRACTEnterococcus faeciumUCN71, isolated from a blood culture, was resistant to low levels of vancomycin (MIC, 16 μg/ml) but susceptible to teicoplanin (MIC, 0.5 μg/ml). No amplification was observed with primers specific for the previously described glycopeptide resistance ligase genes, but a PCR product corresponding to a gene calledvanNwas obtained using degenerate primers and was sequenced. The deduced VanN protein was related (65% identity) to thed-alanine:d-serine VanL ligase. The organization of thevanNgene cluster, determined using degenerate primers and by thermal asymmetric interlaced (TAIL)-PCR, was similar to that of thevanCoperons. A single promoter upstream from the resistance operon was identified by rapid amplification of cDNA ends (RACE)-PCR. The presence of peptidoglycan precursors ending ind-serine andd,d-peptidase activities in the absence of vancomycin indicated constitutive expression of the resistance operon. VanN-type resistance was transferable by conjugation toE. faecium. This is the first report of transferabled-Ala-d-Ser-type resistance inE. faecium.

scholarly journals A New Hypothesis on the Strategy for Acquisition of Phosphorus in Arbuscular Mycorrhiza: Up-Regulation of Secreted Acid Phosphatase Gene in the Host Plant

2005 ◽  
Vol 18 (10) ◽  
pp. 1046-1053 ◽  
Author(s):  
Tatsuhiro Ezawa ◽  
Masahito Hayatsu ◽  
Masanori Saito
Keyword(s):  
Acid Phosphatase ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Mycorrhizal Colonization ◽  
Purple Acid Phosphatase ◽  
Terminal Amino Acid ◽  
Degenerate Primers ◽  
Fungal Partner ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence

The mycorrhiza-responsive phosphatase of Tagetes patula in symbiosis with Glomus etunicatum was detected by electrophoresis, was purified by column chromatography, and was characterized as acid phosphatase that was secreted into rhizosphere. The N-terminal amino acid sequence was determined by a gas-phase sequencer, and a cDNA fragment of the phosphatase gene (TpPAP1) was amplified by degenerate primers designed based on the N-terminal amino acid sequence. The full-length cDNA was obtained by the rapid amplification of cDNA ends technique. The TpPAP1 was of host origin, and the cDNA was 1,843 bp long with a predicted open reading frame of polypeptide of 466 amino acids. Phylogenetic analysis revealed that the gene fell into the cluster of plant high-molecular-weight purple acid phosphatase. Expression analysis of the TpPAP1 in T. patula in symbiosis with Archaeospora leptoticha showed that the levels of transcripts increased eightfold by mycorrhizal colonization. Western blot analysis revealed that the 57-kDa protein corresponding to the mycorrhiza-responsive phosphatase increased by mycorrhizal colonization. The present study proposes a new strategy for acquisition of P in arbuscular mycorrhizal associations in which the fungal partner activates a part of the low-P adaptation system of the plant partner, phosphatase secretion, and improves the overall efficiency of P uptake.

scholarly journals Impact of Elements Containing Glycopeptide Resistance Genes on Expression of Virulence in Enterococcus faecalis Peritonitis: a Pilot Study with Rats

2003 ◽  
Vol 47 (5) ◽  
pp. 1560-1564 ◽  
Author(s):  
G. Plantefeve ◽  
H. Dupont ◽  
V. Hubert ◽  
L. Garry ◽  
C. Poüs ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Resistance Genes ◽  
Peritoneal Fluid ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Glycopeptide Resistance ◽  
Necrosis Factor Alpha ◽  
Cell Counts ◽  
Factor Alpha

ABSTRACT The relationship between virulence and chromosomal elements containing glycopeptide resistance genes was experimentally assessed for two transconjugant strains of Enterococcus faecalis (VanA and VanB phenotypes) and compared to that for a susceptible wild-type strain. Microbiologic and inflammatory effects were assessed in a polymicrobial rat model of peritonitis. Mean peritoneal enterococcus concentrations ± standard deviations at day 1 were 2.1 ± 1.9, 1.3 ± 1.1, and 1.7 ± 2.0 log10 CFU/ml for susceptible, VanA, and VanB strains, respectively (P < 0.05). At day 3 also there were lower concentrations of glycopeptide-resistant enterococcal strains in peritoneal fluid (3.2 ± 3.4, 1.8 ± 1.8, and 2.1 ± 2.4 log10 CFU/ml for susceptible, VanA, and VanB strains, respectively [P < 0.05]). Transconjugant glycopeptide-resistant strains were associated with increased peritoneal cell counts at the different evaluation times of the experiment (P < 0.001). Plasma α1-acid glycoprotein concentrations were lower in the presence of the susceptible strain (667 ± 189 mg/liter) than in the presence of the VanA or VanB strain (1,193 ± 419 or 1,210 ± 404 mg/liter, respectively [P < 0.05]), while concentrations of tumor necrosis factor alpha and interleukin-6 in peritoneal fluid remained similar for the strains. These results suggest a trend toward variation of virulence of transconjugant strains compared to the wild-type strain in this peritonitis model.

scholarly journals Evolutionary conservation of the vertebrate Ah (dioxin) receptor: amplification and sequencing of the PAS domain of a teleost Ah receptor cDNA

1995 ◽  
Vol 310 (2) ◽  
pp. 383-387 ◽  
Author(s):  
M E Hahn ◽  
S I Karchner
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Evolutionary Conservation ◽  
Ah Receptor ◽  
Pas Domain ◽  
Cdna Fragment ◽  
Degenerate Primers ◽  
Reverse Transcription Pcr ◽  
Receptor Cdna ◽  
Dioxin Receptor

The PAS domain of a teleost Ah receptor was amplified using reverse transcription-PCR with degenerate primers containing inosine. The deduced amino acid sequence of the amplified cDNA fragment was 62-64% identical with the PAS domains of mammalian Ah receptors. These data demonstrate the homology of Ah receptors in mammals and fish, and reveal regions of this protein that are highly conserved between these diverse vertebrate groups.

scholarly journals Loss of an individual idiotype on chemical modification. A strategy for assigning idiotypic determinants.

1981 ◽  
Vol 153 (5) ◽  
pp. 1275-1285 ◽  
Author(s):  
J Dickerman ◽  
B Clevinger ◽  
B Friedenson
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Chemical Modification ◽  
Heavy Chain ◽  
Light Chains ◽  
Heavy Chains ◽  
Myeloma Proteins ◽  
Low Levels ◽  
The Individual ◽  
Complementary Strategy

Two dextran-binding myeloma proteins, J558 and Hdex 24, which possess the same individual idiotype (IdI) were diazotized to low levels (1-3.3 groups per subunit) with 1-[14C]-p-aminobenzoate. Both proteins lost the IdI idiotype under these conditions with most of the label incorporated on the heavy chains of each protein. When the diazotization ws carried out in the presence of the hapten 1-O-methyl-alpha-D-glucopyranoside the loss of idiotypic reactivity could be prevented for J558 but not for Hdex 24. Under these conditions most of the label was incorporated on the light chains of J558, but on the heavy chains of Hdex 24. For J558, these results show that a major determinant of the individual idiotype is within the hypervariable positions of the heavy chain. For Hdex 24 the determinant being modified is on the heavy chain but not involved in hapten binding. These results are consistent with previous work showing that J558 and Hdex 24 differ in amino acid sequence in the D and the J segments of the heavy chain and offer an alternative and complementary strategy for assigning idiotypic determinants.

scholarly journals Protein structure and gene cloning of Syncephalastrum racemosum nuclease

1999 ◽  
Vol 339 (2) ◽  
pp. 261-267 ◽  
Author(s):  
Heng-Chien HO ◽  
Ta-Hsiu LIAO
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site ◽  
Direct Sequencing ◽  
Coli Strain ◽  
Protein Sequencing ◽  
Intact Protein ◽  
Computer Search ◽  
Degenerate Primers ◽  
Syncephalastrum Racemosum

The complete amino acid sequence of the fungus Syncephalastrum racemosum (Sr-) nuclease has been delineated on the basis of protein sequencing of the intact protein and its protease-digested peptides. The resulting 250-residue sequence shows a carbohydrate side chain attached at Asn134 and two half-cystine residues (Cys242 and Cys247) cross-linked to form a small disulphide loop. On the basis of the sequence of Sr-nuclease, a computer search in the sequence database yielded 60% and 48% positional identities with the sequences of Cunninghamella echinulata nuclease C1 and yeast mitochondria nuclease respectively, and very little similarity to those of several known mammalian DNases I. Sequence alignment of the three similar nucleases reveals that the single small disulphide loop is unchanged but the carbohydrate attachment in Sr-nuclease is absent from the other two nucleases. Alignment also shows a highly conserved region harbouring Sr-nuclease His85, which is assigned as one of the essential residues in the active site. The cDNA encoding Sr-nuclease was amplified by using reverse transcriptase-mediated PCR with degenerate primers based on its amino acid sequence. Subsequently, specific primers were synthesized for use in the 3´ and 5´ rapid amplification of cDNA ends (RACE). Direct sequencing of the RACE products led to the deduction of a 1.1 kb cDNA sequence for Sr-nuclease. The cDNA contains an open reading frame of 320 amino acid residues including a 70-residue putative signal peptide and the 250-residue mature protein. Finally, the recombinant Sr-nuclease was expressed in Escherichia coli strain BL21(DE3) in which the recombinant protein, after solubilization with detergent and renaturation, showed both DNase and RNase activities. The assignment of His85 to the active site was further supported by evidence that the mutant protein Sr-nuclease (H85A), in which His85 was replaced by Ala, was not able to degrade DNA or RNA.

scholarly journals Molecular Characterization of a Novel Glucosyltransferase from Lactobacillus reuteri Strain 121 Synthesizing a Unique, Highly Branched Glucan with α-(1→4) and α-(1→6) Glucosidic Bonds

2002 ◽  
Vol 68 (9) ◽  
pp. 4283-4291 ◽  
Author(s):  
S. Kralj ◽  
G. H. van Geel-Schutten ◽  
H. Rahaoui ◽  
R. J. Leer ◽  
E. J. Faber ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Characterization ◽  
Lactobacillus Reuteri ◽  
Degenerate Primers ◽  
Terminal Domain ◽  
Branching Points ◽  
Culture Supernatants

ABSTRACT Lactobacillus reuteri strain 121 produces a unique, highly branched, soluble glucan in which the majority of the linkages are of the α-(1→4) glucosidic type. The glucan also contains α-(1→6)-linked glucosyl units and 4,6-disubstituted α-glucosyl units at the branching points. Using degenerate primers, based on the amino acid sequences of conserved regions from known glucosyltransferase (gtf) genes from lactic acid bacteria, the L. reuteri strain 121 glucosyltransferase gene (gtfA) was isolated. The gtfA open reading frame (ORF) was 5,343 bp, and it encodes a protein of 1,781 amino acids with a deduced M r of 198,637. The deduced amino acid sequence of GTFA revealed clear similarities with other glucosyltransferases. GTFA has a relatively large variable N-terminal domain (702 amino acids) with five unique repeats and a relatively short C-terminal domain (267 amino acids). The gtfA gene was expressed in Escherichia coli, yielding an active GTFA enzyme. With respect to binding type and size distribution, the recombinant GTFA enzyme and the L. reuteri strain 121 culture supernatants synthesized identical glucan polymers. Furthermore, the deduced amino acid sequence of the gtfA ORF and the N-terminal amino acid sequence of the glucosyltransferase isolated from culture supernatants of L. reuteri strain 121 were the same. GTFA is thus responsible for the synthesis of the unique glucan polymer in L. reuteri strain 121. This is the first report on the molecular characterization of a glucosyltransferase from a Lactobacillus strain.

Sign in / Sign up

Export Citation Format

Share Document