Article (Scientific journals)
Transcription and expression analysis, using lacZ and phoA gene fusions, of Mycobacterium fortuitum beta-lactamase genes cloned from a natural isolate and a high-level beta-lactamase producer.
Timm, J; Perilli, M G; Duez, Colette et al.
1994In Molecular Microbiology, 12 (3), p. 491-504
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Keywords :
Alkaline Phosphatase/biosynthesis/genetics; Amino Acid Sequence; Ampicillin Resistance/genetics; Base Sequence; Cloning, Molecular; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Genes, Bacterial/genetics; Molecular Sequence Data; Mycobacterium/enzymology/genetics; Promoter Regions, Genetic/genetics; Recombinant Fusion Proteins/biosynthesis; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; beta-Galactosidase/biosynthesis/genetics; beta-Lactamases/physiology
Abstract :
[en] The gene encoding a class A beta-lactamase was cloned from a natural isolate of Mycobacterium fortuitum (blaF) and from a high-level amoxicillin-resistant mutant that produces large amounts of beta-lactamase (blaF*). The nucleotide sequences of the two genes differ at 11 positions, including two in the region upstream from the coding sequence. Gene fusions to Escherichia coli lacZ and transcription and expression analysis of the cloned genes in Mycobacterium smegmatis indicated that high-level production of the beta-lactamase in the mutant is mainly or wholly due to a single base pair difference in the promoter. These analyses also showed that transcription and translation start at the same position. A comparison of the amino acid sequence of BlaF, as predicted from the nucleotide sequence, with the determined N-terminal amino acid sequence indicated the presence of a typical signal peptide. The fusion of blaF (or blaF*) to the E. coli gene phoA resulted in the production of BlaF-PhoA hybrid proteins that had alkaline phosphatase activity. These results demonstrate that phoA can be used as a reporter gene for studying protein export in mycobacteria.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Timm, J
Perilli, M G
Duez, Colette ;  Université de Liège - ULiège > Centre d'ingénierie des protéines
Trias, J
Orefici, G
Fattorini, L
Amicosante, G
Oratore, A
Joris, Bernard ;  Université de Liège - ULiège > Département des sciences de la vie > Physiologie et génétique bactériennes
Frère, Jean-Marie ;  Université de Liège - ULiège > Centre d'ingénierie des protéines
Language :
English
Title :
Transcription and expression analysis, using lacZ and phoA gene fusions, of Mycobacterium fortuitum beta-lactamase genes cloned from a natural isolate and a high-level beta-lactamase producer.
Publication date :
1994
Journal title :
Molecular Microbiology
ISSN :
0950-382X
eISSN :
1365-2958
Publisher :
Blackwell Publishing, Oxford, United Kingdom
Volume :
12
Issue :
3
Pages :
491-504
Peer reviewed :
Peer Reviewed verified by ORBi
Available on ORBi :
since 22 May 2012

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