Characterization of gentamicin 2'-N-acetyltransferase from Providencia stuartii: its use of peptidoglycan metabolites for acetylation of both aminoglycosides and peptidoglycan

Payie, Kenneth G.; Clarke, Anthony J.

doi:10.1128/jb.179.13.4106-4114.1997

Cited by 36 publications

(35 citation statements)

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“…Integrons could "mop" up these genes following excision, and incorporate them into replicons, which could then be transferred among bacterial pathogens. Several pieces of evidence similarly support the hypothesis that housekeeping genes have evolved to modify antibiotics and that their recruitment could provide an alternate source of resistance genes [23,68]. All resistance-encoding DNAs establish a resistance gene pool, which represents a potential source of gene cassettes incorporated into the integrons [28].…”

Section: Mobile Gene Cassettesmentioning

confidence: 93%

Importance of integrons in the diffusion of resistance

2001

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Summary -Horizontal transfer of resistance genes is a successful mechanism for the transmission and dissemination of multiple drug resistance among bacterial pathogens. The impact of horizontally transmitted genetic determinants in the evolution of resistance is particularly evident when resistance genes are physically associated in clusters and transferred en bloc to the recipient cell. Recent advances in the molecular characterisation of antibiotic resistance mechanisms have highlighted the existence of genetic structures, called integrons, involved in the acquisition of resistance genes. These DNA elements have frequently been reported in multi-drug resistant strains isolated from animals and humans, and are located either on the bacterial chromosome or on broad-host-range plasmids. The role of integrons in the development of multiple resistance relies on their unique capacity to cluster and express drug resistance genes. Moreover, the spread of resistance genes among different replicons and their exchange between plasmid and bacterial chromosome are facilitated by the integration of integrons into transposable elements. The association of a highly efficient gene capture and expression system, together with the capacity for vertical and horizontal transmission of resistance genes represents a powerful weapon used by bacteria to combat the assault of antibiotics. antimicrobial resistance / integron / transposon / horizontal gene transferRésumé -Importance des intégrons dans la diffusion de la résistance. Le transfert horizontal de gènes est un mécanisme efficace pour la transmission et la dissémination, parmi les agents pathogènes bactériens, de résistances multiples aux antibiotiques. L'impact des déterminants génétiques transmis horizontalement dans l'évolution de la résistance est particulièrement évident lorsque les gènes de résistance sont physiquement regroupés, et transférés en bloc à la cellule réceptrice. Des avancées récentes dans la caractérisation moléculaire des mécanismes de résistance aux antibiotiques ont mis en évidence l'existence de structures génétiques, appelées intégrons, impliqués dans l'acquisition de gènes de résistance. Ces éléments d'ADN ont été fréquemment observés dans des souches bactériennes multi-résistantes isolées chez des animaux ou chez des humains, localisés soit sur le chromosome bactérien, soit sur des plasmides à large spectre d'hôte. Le rôle des intégrons dans le développement de la multi-résistance repose sur leur capacité unique à former des clusters de gènes et à exprimer des gènes Vet. Res. 32 (2001) 243-259 243

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Section: Mobile Gene Cassettesmentioning

confidence: 93%

Importance of integrons in the diffusion of resistance

2001

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“…Even though the natural substrate(s) for Eis are unknown, the protein could be also involved in metabolic activities of the cell. Aminoglycoside acetyltransferases have been hypothesized to play a role in metabolism (25), although the only clear example of a metabolic function for one of these proteins is the O-acetylation of peptidoglycan in Providencia stuartii (26,27).…”

Section: Discussionmentioning

confidence: 99%

Overexpression of the chromosomally encoded aminoglycoside acetyltransferase eis confers kanamycin resistance in Mycobacterium tuberculosis

Zaunbrecher

Sikes²,

Metchock³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

300

311

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The emergence of multidrug-resistant (MDR) tuberculosis (TB) highlights the urgent need to understand the mechanisms of resistance to the drugs used to treat this disease. The aminoglycosides kanamycin and amikacin are important bactericidal drugs used to treat MDR TB, and resistance to one or both of these drugs is a defining characteristic of extensively drug-resistant TB. We identified mutations in the ؊10 and ؊35 promoter region of the eis gene, which encodes a previously uncharacterized aminoglycoside acetyltransferase. These mutations led to a 20 -180-fold increase in the amount of eis leaderless mRNA transcript, with a corresponding increase in protein expression. Importantly, these promoter mutations conferred resistance to kanamycin [5 g/mL < minimum inhibitory concentration (MIC) <40 g/mL] but not to amikacin (MIC <4 g/mL). Additionally, 80% of clinical isolates examined in this study that exhibited low-level kanamycin resistance harbored eis promoter mutations. These results have important clinical implications in that clinical isolates determined to be resistant to kanamycin may not be cross-resistant to amikacin, as is often assumed. Molecular detection of eis mutations should distinguish strains resistant to kanamycin and those resistant to kanamycin and amikacin. This may help avoid excluding a potentially effective drug from a treatment regimen for drug-resistant TB.T he World Health Organization estimates that 9.2 million new cases of tuberculosis (TB) occur each year (1). Despite intensive efforts to ensure proper drug dosages and patient compliance with drug regimens, multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis have emerged (2). These strains cause extensive mortality in immunocompromised individuals (3) and hinder the control and prevention of the disease. XDR and MDR TB infections cannot be adequately treated with the first-line anti-TB drugs and require expensive, prolonged treatment with second-line anti-TB drugs. The rapid determination of the resistance profile of an isolate can facilitate selection of an appropriate drug regimen and preclude development of additional drug resistances. Rapid detection of resistances is best achieved with molecular diagnostic approaches, particularly in developing countries where access to culture facilities is limited. Such strategies require a detailed understanding of the molecular basis for drug resistance. Although the mechanisms of resistance to first-line drugs such as isoniazid and rifampin are well characterized, much less is known about such mechanisms for the second-line drugs (4).An important second-line anti-TB drug is the aminoglycoside kanamycin (KAN), which binds to the 16S rRNA in the 30S ribosomal subunit and inhibits protein synthesis (5). In other bacteria, characterized mechanisms of KAN resistance include altered efflux or influx of the drug, inactivation of the drug by enzymatic modification, and mutation or methylation of rRNA, which disrupts binding of the drug to the riboso...

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“…It seems unlikely this L. pneumophila enzyme has evolved specifically for the detoxification of Spec, and this reaction may be fortuitous, although the ecology of organism is not well known and it may come into contact with Spec-producing organisms. Perhaps, like the aminoglycoside acetyltransferase AAC(2Ј)-Ia from Providencia stuartii where this enzyme is thought to play a role in peptidoglycan modification (28), APH(9)-Ia may also have an as yet undetermined physiological role phosphorylating other substrates important to L. pneumophila.…”

Section: Implications and Conclusionmentioning

confidence: 99%

Spectinomycin Kinase from Legionella pneumophila

Thompson

Hughes

Cianciotto

et al. 1998

Journal of Biological Chemistry

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The bacterium Legionella pneumophila is the responsible agent for Legionnaires' disease and has recently been shown to harbor a gene encoding a kinase that confers resistance to the aminoglycoside antibiotic spectinomycin (Suter, T. M., Viswanathan, V. K., and Cianciotto, N. P. (1997) Antimicrob. Agents Chemother. 41, 1385-1388). We report the overproduction, purification, and characterization of this spectinomycin kinase from an expressing system in Escherichia coli. The purified protein shows stringent substrate specificity for spectinomycin with K m ‫؍‬ 21.5 M and k cat ‫؍‬ 24.2 s ؊1 and does not bind other aminoglycosides including kanamycin, amikacin, neomycin, butirosin, streptomycin, or apramycin. Purification of spectinomycin phosphate followed by characterization by mass spectrometry and 1 H, 13 C, and 31 P NMR established the site of phosphorylation to be at the hydroxyl group at position 9. Thus this enzyme is designated APH(9)-Ia (where APH is aminoglycoside kinase). The enzyme was inactivated by the electrophilic ATP analogue 5-[p-(fluorosulfonyl)benzoyl]adenosine, consistent with a nucleophilic residue such as Lys lining the nucleotide binding pocket. Sitedirected mutagenesis of Lys-52 and Asp-212 to Ala confirmed that these residues were important for catalysis, with Lys-52 playing a potential role in ATP binding and Asp-212 in phosphoryl transfer. Thio and solvent isotope effect experiments in the presence of either Mg 2؉ or Mn 2؉ were consistent with a kinetic mechanism in which phosphate transfer does not contribute significantly to the rate-limiting step. These results establish that APH(9)-Ia is a highly specific antibiotic resistance kinase and provides the requisite mechanistic information for future structural studies.

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Characterization of gentamicin 2'-N-acetyltransferase from Providencia stuartii: its use of peptidoglycan metabolites for acetylation of both aminoglycosides and peptidoglycan

Cited by 36 publications

References 50 publications

Importance of integrons in the diffusion of resistance

Importance of integrons in the diffusion of resistance

Overexpression of the chromosomally encoded aminoglycoside acetyltransferase eis confers kanamycin resistance in Mycobacterium tuberculosis

Spectinomycin Kinase from Legionella pneumophila

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