The gene encoding a class A β-lactamase was cloned from a natural isolate of Mycobacterium fortuitum (blaF) and from a high-level amoxicillin-resistant mutant that produces large amounts of β-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 β- 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.

Transcription and expression analysis, using lacZ and phoA gene fusions, of Mycobacterium fortuitum β-lactamase genes cloned from a natural isolate and a high-level β-lactamase producer

AMICOSANTE, Gianfranco;ORATORE, Arduino;
1994-01-01

Abstract

The gene encoding a class A β-lactamase was cloned from a natural isolate of Mycobacterium fortuitum (blaF) and from a high-level amoxicillin-resistant mutant that produces large amounts of β-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 β- 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.
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11697/103124
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus 79
  • ???jsp.display-item.citation.isi??? 88
social impact