Browse
Submit Data
Databases
API
Help

Dataset Information

3 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Cloning, sequencing, and use as a molecular probe of a gene encoding an aminoglycoside 6'-N-acetyltransferase of broad substrate profile.

ABSTRACT: A gene coding for an aminoglycoside 6'-N-acetyltransferase that was able to modify amikacin was cloned from a plasmid isolated from a clinical strain of Enterobacter cloacae. Sequencing of a 955-bp segment which mediates the modifying activity revealed a single open reading frame of 432 nucleotides that predicted a polypeptide of 144 amino acid residues with a molecular weight of 16,021. Putative ribosomal binding sites and -10 and -35 sequences were located at the 5' end of the gene. The size of the polypeptide was confirmed through minicell analysis of the expression products of plasmids containing the sequence. The use of the gene as a molecular probe revealed its specificity toward strains harboring genes coding for related enzymes. This probe is therefore useful for epidemiological studies.

SUBMITTER: Teran FJ

PROVIDER: S-EPMC245084 | biostudies-other | 1991 Apr

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Publications

Cloning, sequencing, and use as a molecular probe of a gene encoding an aminoglycoside 6'-N-acetyltransferase of broad substrate profile.

Terán F J FJ Suárez J E JE Mendoza M C MC

Antimicrobial agents and chemotherapy 19910401 4

A gene coding for an aminoglycoside 6'-N-acetyltransferase that was able to modify amikacin was cloned from a plasmid isolated from a clinical strain of Enterobacter cloacae. Sequencing of a 955-bp segment which mediates the modifying activity revealed a single open reading frame of 432 nucleotides that predicted a polypeptide of 144 amino acid residues with a molecular weight of 16,021. Putative ribosomal binding sites and -10 and -35 sequences were located at the 5' end of the gene. The size o ...[more]

PMID: 2069376

Similar Datasets

Aminoglycoside resistance profile and structural architecture of the aminoglycoside acetyltransferase AAC(6')-Im.

Project description:Aminoglycoside 6'-acetyltransferase-Im (AAC(6')-Im) is the closest monofunctional homolog of the AAC(6')-Ie acetyltransferase of the bifunctional enzyme AAC(6')-Ie/APH(2")-Ia. The AAC(6')-Im acetyltransferase confers 4- to 64-fold higher MICs to 4,6-disubstituted aminoglycosides and the 4,5-disubstituted aminoglycoside neomycin than AAC(6')-Ie, yet unlike AAC(6')-Ie, the AAC(6')-Im enzyme does not confer resistance to the atypical aminoglycoside fortimicin. The structure of the kanamycin A complex of AAC(6')-Im shows that the substrate binds in a shallow positively-charged pocket, with the N6' amino group positioned appropriately for an efficient nucleophilic attack on an acetyl-CoA cofactor. The AAC(6')-Ie enzyme binds kanamycin A in a sufficiently different manner to position the N6' group less efficiently, thereby reducing the activity of this enzyme towards the 4,6-disubstituted aminoglycosides. Conversely, docking studies with fortimicin in both acetyltransferases suggest that the atypical aminoglycoside might bind less productively in AAC(6')-Im, thus explaining the lack of resistance to this molecule.

| S-EPMC5722643 | biostudies-literature

New integron-associated gene cassette encoding a 3-N-aminoglycoside acetyltransferase.

Project description:A fifth gene cassette containing an aacC gene, aacCA5, was found in an aacCA5-aadA7 cassette array in a class 1 integron isolated from a multiply drug resistant Salmonella enterica serovar Kentucky strain. The AacC-A5 or AAC(3)-Ie acetyltransferase encoded by aacCA5 is related to other AAC(3)-I enzymes and confers resistance to gentamicin.

| S-EPMC549260 | biostudies-literature

Synthesis of 4'-aminopantetheine and derivatives to probe aminoglycoside N-6'-acetyltransferase.

Project description:A convenient synthesis of 4'-aminopantetheine from commercial D-pantethine is reported. The amino group was introduced by reductive amination in order to avoid substitution at a sterically congested position. Derivatives of 4'-aminopantetheine were also prepared to evaluate the effect of O-to-N substitution on inhibitors of the resistance-causing enzyme aminoglycoside N-6'-acetyltransferase. The biological results combined with docking studies indicate that in spite of its reported unusual flexibility and ability to adopt different folds, this enzyme is highly specific for AcCoA.

| S-EPMC3084192 | biostudies-literature

Aminoglycoside 6'-N-acetyltransferase variants of the Ib type with altered substrate profile in clinical isolates of Enterobacter cloacae and Citrobacter freundii.

Project description:Three clinical isolates, Enterobacter cloacae EC1562 and EC1563 and Citrobacter freundii CFr564, displayed an aminoglycoside resistance profile evocative of low-level 6'-N acetyltransferase type II [AAC(6')-II] production, which conferred reduced susceptibility to gentamicin but not to amikacin or isepamicin. Aminoglycoside acetyltransferase assays suggested the synthesis in the three strains of an AAC(6') which acetylated amikacin practically as well as it acetylated gentamicin in vitro. Both compounds, however, as well as isepamicin, retained good bactericidal activity against the three strains. The aac genes were borne by conjugative plasmids (pLMM562 and pLMM564 of ca. 100 kb and pLMM563 of ca. 20 kb). By PCR mapping and nucleotide sequence analysis, an aac(6')-Ib gene was found in each strain upstream of an ant(3")-I gene in a sulI-type integron. The size of the AAC(6')-Ib variant encoded by pLMM562 and pLMM564, AAC(6')-Ib7, was deduced to be 184 (or 177) amino acids long, whereas in pLMM563 a 21-bp duplication allowing the recruitment of a start codon resulted in the translation of a variant, AAC(6')-Ib8, of 196 amino acids, in agreement with size estimates obtained by Western blot analysis. Both variants had at position 119 a serine instead of the leucine typical for the AAC(6')-Ib variants conferring resistance to amikacin. By using methods that predict the secondary structure, these two amino acids appear to condition an alpha-helical structure within a putative aminoglycoside binding domain of AAC(6')-Ib variants.

| S-EPMC105389 | biostudies-literature

Cloning, sequencing and heterologous expression of a cDNA encoding pigeon liver carnitine acetyltransferase.

Project description:Two overlapping cDNA clones encoding pigeon liver carnitine acetyltransferase (EC 2.3.1.7) (CAT) were isolated from a pigeon liver lambda gt11 cDNA library by gene amplification using oligonucleotide primers based on the N-terminal amino acid sequence of the enzyme. The two clones, which represent the 5' and 3' ends of the gene, were spliced together to form a single cDNA construct containing the entire coding sequence for CAT, with an in-frame TGA stop codon 42 bases before the first ATG start site and a 3'-untranslated segment of 1057 bases. The largest open reading frame of 1942 nucleotides predicted a polypeptide of 627 amino acids and a molecular mass of 71.1 kDa. The N-terminus and four internal peptides from the amino acid sequence of pigeon breast muscle CAT were identified in the predicted sequence of the liver cDNA clone. The identity of the CAT cDNA was confirmed by heterologous expression of active recombinant CAT (rCAT) in insect cells using the baculovirus expression system. Western blots of rCAT from infected insect cell lysates and immunodetection with a rabbit anti-CAT polyclonal serum showed an immunoreactive protein band similar in size to native CAT from pigeon breast muscle. Like the native enzyme, rCAT was capable of acylating carnitine with a preference for small-chain acyl-CoAs of carbon chain lengths C2-C4.

| S-EPMC1136381 | biostudies-other

Synthesis and use of sulfonamide-, sulfoxide-, or sulfone-containing aminoglycoside-CoA bisubstrates as mechanistic probes for aminoglycoside N-6'-acetyltransferase.

Project description:Aminoglycoside-coenzyme A conjugates are challenging synthetic targets because of the wealth of functional groups and high polarity of the starting materials. We previously reported a one-pot synthesis of amide-linked aminoglycoside-CoA bisubstrates. These molecules are nanomolar inhibitors of aminoglycoside N-6'-acetyltransferase Ii (AAC(6')-Ii), an important enzyme involved in bacterial resistance to aminoglycoside antibiotics. We report here the synthesis and biological activity of five new aminoglycoside-CoA bisubstrates containing sulfonamide, sulfoxide, or sulfone groups. Interestingly, the sulfonamide-linked bisubstrate, which was expected to best mimic the tetrahedral intermediate, does not show improved inhibition when compared with amide-linked bisubstrates. On the other hand, most of the sulfone- and sulfoxide-containing bisubstrates prepared are nanomolar inhibitors of AAC(6')-Ii.

| S-EPMC3084191 | biostudies-literature

Integron-Derived Aminoglycoside-Sensing Riboswitches Control Aminoglycoside Acetyltransferase Resistance Gene Expression.

Project description:Class 1 integrons accumulate antibiotic resistance genes by site-specific recombination at aatI-1 sites. Captured genes are transcribed from a promoter located within the integron; for class 1 integrons, the first gene to be transcribed and translated normally encodes an aminoglycoside antibiotic resistance protein (either an acetyltransferase [AAC] or adenyltransferase [AAD]). The leader RNA from the Pseudomonas fluorescens class 1 integron contains an aminoglycoside-sensing riboswitch RNA that controls the expression of the downstream aminoglycoside resistance gene. Here, we explore the relationship between integron-dependent DNA recombination and potential aminoglycoside-sensing riboswitch products of recombination derived from a series of aminoglycoside-resistant clinical strains. Sequence analysis of the clinical strains identified a series of sequence variants that were associated with class I integron-derived aminoglycoside-resistant (both aac and aad) recombinants. For the aac recombinants, representative sequences showed up to 6-fold aminoglycoside-dependent regulation of reporter gene expression. Microscale thermophoresis (MST) confirmed RNA binding. Covariance analysis generated a secondary-structure model for the RNA that is an independent verification of previous models that were derived from mutagenesis and chemical probing data and that was similar to that of the P. fluorescens riboswitch RNA. The aminoglycosides were among the first antibiotics to be used clinically, and the data suggest that in an aminoglycoside-rich environment, functional riboswitch recombinants were selected during integron-mediated recombination to regulate aminoglycoside resistance. The incorporation of a functional aminoglycoside-sensing riboswitch by integron recombination confers a selective advantage for the expression of resistance genes of diverse origins.

| S-EPMC6535548 | biostudies-literature

Structural analysis of a novel substrate-free form of the aminoglycoside 6'-N-acetyltransferase from Enterococcus faecium.

Project description:Aminoglycoside acetyltransferases (AACs) catalyze the transfer of an acetyl group between acetyl-CoA and an aminoglycoside, producing CoA and an acetylated aminoglycoside. AAC(6')-Ii enzymes target the amino group linked to the 6' C atom in an aminoglycoside. Several structures of the AAC(6')-Ii from Enterococcus faecium [Ef-AAC(6')-Ii] have been reported to date. However, the detailed mechanism of its enzymatic function remains elusive. In this study, the crystal structure of Ef-AAC(6')-Ii was determined in a novel substrate-free form. Based on structural analysis, it is proposed that Ef-AAC(6')-Ii sequentially undergoes conformational selection and induced fit for substrate binding. These results therefore provide a novel viewpoint on the mechanism of action of Ef-AAC(6')-Ii.

| S-EPMC7397467 | biostudies-literature

Cloning and characterization of a 3-N-aminoglycoside acetyltransferase gene, aac(3)-Ib, from Pseudomonas aeruginosa.

Project description:A novel gene encoding an aminoglycoside 3-N-acetyltransferase, which confers resistance to gentamicin, astromicin, and sisomicin, was cloned from Pseudomonas aeruginosa Stone 130. Its sequence was determined and found to show considerable similarity to an aac(3)-I gene previously cloned from R plasmids from Enterobacter, Pseudomonas, and Serratia spp. We have designated the genes from the R plasmids and this work aac(3)-Ia and aac(3)-Ib, respectively. The two aac(3)-I genes share 74% nucleotide identity, and their deduced protein products are 88% similar. These data suggest that the genes derive from a common ancestor. Homology between the flanking sequences of both aac(3)-I genes and other resistance determinants known to reside in integron environments was also observed. Intragenic probes specific for either aac(3)-Ia or aac(3)-Ib were used in hybridization studies with a series of gentamicin-, astromicin-, and sisomicin-resistant clinical isolates. Of 59 clinical isolates tested, no isolates hybridized with both probes, 30 (51%) hybridized with the aac(3)-Ia probe, 12 (20%) hybridized with the aac(3)-Ib probe, and 17 (29%) did not hybridize with either probe. These data suggest the existence of at least one other aac(3)-I gene.

| S-EPMC162827 | biostudies-other

Characterization of the aac(6')-Ib gene encoding an aminoglycoside 6'-N-acetyltransferase in Pseudomonas aeruginosa BM2656.

Project description:Pseudomonas aeruginosa BM2656 was resistant to tobramycin and susceptible to gentamicin and amikacin by disk diffusion testing. This unusual resistance was not transferable by conjugation to Escherichia coli or P. aeruginosa PAO38, and plasmid DNA was not detected in this strain. A 0.9-kb fragment harboring the tobramycin resistance gene was cloned from BM2656 into pUC18, generating pAT129. Analysis for aminoglycoside-modifying activity in extracts of BM2656 and E. coli harboring pAT129 indicated that tobramycin resistance was due to synthesis of an aminoglycoside 6'-N-acetyltransferase type I [AAC(6')-I] enzyme which modified amikacin and tobramycin. Although amikacin was acetylated, the bactericidal synergism of this aminoglycoside with ceftazidime against BM2656 was minimally affected. The sequence of the DNA fragment was determined. It contained an aac (6')-Ib-like gene and was located downstream from a conserved region related to Tn21. The translated sequence of this aac(6')-Ib gene possessed 99.2% identity with the putative products of the aac(6')-Ib gene cassettes from Serratia marcescens and Klebsiella pneumoniae and 69% identity with the putative aacA(6')-II gene product from P. aeruginosa. We conclude that an aac(6')-Ib gene has spread to the chromosome of P. aeruginosa, probably by transposition.

| S-EPMC187994 | biostudies-other

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data