PMID- 10627047
OWN - NLM
STAT- MEDLINE
DCOM- 20000217
LR  - 20200826
IS  - 1350-0872 (Print)
IS  - 1350-0872 (Linking)
VI  - 145 ( Pt 12)
DP  - 1999 Dec
TI  - Production of mutants in amino acid biosynthesis genes of Mycobacterium 
      tuberculosis by homologous recombination.
PG  - 3497-3503
LID - 10.1099/00221287-145-12-3497 [doi]
AB  - The ability to generate mutants of Mycobacterium tuberculosis will be important 
      if we are to understand the biology of this major pathogen. However, allelic 
      replacement methods have only recently achieved success. We have developed a 
      reproducible method for generating defined mutants of M. tuberculosis using 
      homologous recombination. The transforming DNA was used following pre-treatment 
      either with UV light or alkali denaturation in order to stimulate homologous 
      recombination and abolish illegitimate recombination. Suicide vectors carrying 
      one of nine amino acid biosynthesis genes were electroporated into M. 
      tuberculosis, and homologous recombinants were obtained in all nine genes; eight 
      resulted from single-crossover events (SCOs) and one from a double-crossover 
      event (DCO) (in the metB gene). The remaining colonies were spontaneous 
      hygromycin-resistant mutants; no products of illegitimate recombination were 
      observed. To more efficiently distinguish spontaneous mutants, the lacZ gene was 
      cloned into five vectors (two containing genes not previously tested), and the 
      transformations were repeated. SCO mutants were identified by screening for blue 
      colonies on indicator plates. White transformants were tested for auxotrophy and 
      trpD, hisD and proC auxotrophic mutants were obtained. Only blue SCOs were 
      obtained for argF and glnE. Thus, using this methodology we have obtained 
      homologous recombination in 11 genes, and DCOs in 4 genes, showing that it is 
      possible to generate targeted mutants in a reproducible way.
FAU - Parish, Tanya
AU  - Parish T
AD  - Department of Infectious and Tropical Diseases, London School of Hygiene & 
      Tropical Medicine, Keppel Street, London WC1E 7HT, UK 1.
FAU - Gordhan, Bhavna G
AU  - Gordhan BG
AD  - Molecular Biology Unit, South African Institute for Medical Research, PO Box 
      1038, Johannesburg 2000, South Africa 2.
FAU - McAdam, Ruth A
AU  - McAdam RA
AD  - Immunopathology Unit, Glaxo Wellcome Research and Development, Medicines Research 
      Centre, Stevenage, UK3.
FAU - Duncan, Ken
AU  - Duncan K
AD  - Immunopathology Unit, Glaxo Wellcome Research and Development, Medicines Research 
      Centre, Stevenage, UK3.
FAU - Mizrahi, Valerie
AU  - Mizrahi V
AD  - Department of Haematology and Molecular Medicine, University of the 
      Witwatersrand, Johannesburg, South Africa4.
AD  - Molecular Biology Unit, South African Institute for Medical Research, PO Box 
      1038, Johannesburg 2000, South Africa 2.
FAU - Stoker, Neil G
AU  - Stoker NG
AD  - Department of Infectious and Tropical Diseases, London School of Hygiene & 
      Tropical Medicine, Keppel Street, London WC1E 7HT, UK 1.
LA  - eng
GR  - Wellcome Trust/United Kingdom
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - England
TA  - Microbiology (Reading)
JT  - Microbiology (Reading, England)
JID - 9430468
RN  - 0 (Amino Acids)
RN  - 0 (DNA, Bacterial)
SB  - IM
MH  - Amino Acids/*biosynthesis/genetics
MH  - Crossing Over, Genetic
MH  - DNA, Bacterial/genetics/radiation effects
MH  - Electroporation/methods
MH  - Genetic Vectors
MH  - Hydrogen-Ion Concentration
MH  - Lac Operon
MH  - *Mutation
MH  - Mycobacterium tuberculosis/*genetics/growth & development/*metabolism
MH  - Plasmids/genetics
MH  - *Recombination, Genetic
MH  - Transformation, Bacterial
MH  - Ultraviolet Rays
EDAT- 2000/01/08 09:00
MHDA- 2000/02/19 09:00
CRDT- 2000/01/08 09:00
PHST- 2000/01/08 09:00 [pubmed]
PHST- 2000/02/19 09:00 [medline]
PHST- 2000/01/08 09:00 [entrez]
AID - 10.1099/00221287-145-12-3497 [doi]
PST - ppublish
SO  - Microbiology (Reading). 1999 Dec;145 ( Pt 12):3497-3503. doi: 
      10.1099/00221287-145-12-3497.