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Title: Gentamicin resistance in Micromonospora purpurea
Authors: Bacon, Joanna.
Award date: 1999
Presented at: University of Leicester
Abstract: The gene, grmA, from the gentamicin producer Micromonospora purpurea, confers high level resistance to gentamicin and kanamycin. The ribosomes of M. purpurea are constitutively resistant to these antibiotics by modification of the 16S rRNA in the 30S subunit.;grmA encodes a methyltransferase. In this study, reverse transcriptase assays confirmed that GrmA methylated the 16S rRNA at residue G1389 in S. lividans. However these assays also revealed that G1389 was not methylated in M. purpurea, indicating that grmA is silent in its native host.;grmA, driven by a constitutive promoter (ermEp*), was introduced into M. purpurea by conjugal transfer with the hope that the effects of a putative repressor would be titrated out by the expression of an extra copy of the gene. Changes in the level of grmA transcript and methylation of the 18S rRNA were subsequently observed.;RNA hybridisation analysis revealed that grmA was transcribed in the wild type strain of M. purpurea, and that the transcript was probably full length. There was a higher level of grmA transcript in S. lividans containing the gene than in M. purpurea, suggesting that grmA was transcriptionally regulated in M. purpurea but not in S. lividans.;The extra copy of grmA in M. purpurea was expressed efficiently from ermEp*. However, 16S rRNA extracted from this strain, had not been methylated at G1389, although the transcriptional control of grmA was apparently alleviated. These data suggested that there may have been more than one control mechanism regulating the expression of grmA.;If grmA is not expressed in M. purpurea, there must be another resistance gene responsible for the constitutive phenotype being expressed. Despite attempts to isolate alternative gentamicin resistance genes from M. purpurea, only grmA was found.
Type: Thesis
Level: Doctoral
Qualification: PhD
Rights: Copyright © the author. All rights reserved.
Appears in Collections:Theses, Dept. of Biochemistry
Leicester Theses

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