Related Experiment Videos
Plasmid-mediated NAD independence in Haemophilus parainfluenzae
H M Windsor1, R C Gromkova, H J Koornhof
1Emergent Pathogen Research Unit, South African Medical Research Council, School of Pathology, University of the Witwatersrand.
Abstract:
The location of the genes coding for NAD independence in four unusual clinical isolates of Haemophilus parainfluenzae was determined by transferring these genes to plasmid-free Haemophilus influenzae Rd by transformation and analysing transformants for the presence of plasmids by agarose gel electrophoresis. All NAD-independent transformants were found to carry a single plasmid species. The plasmids, originally harboured by the four H. parainfluenzae isolates recovered from unrelated sources, were of the same size (5.25 kb). Spontaneous reversion to NAD dependence occurred with a low frequency (0.1 to 0.2% of the progeny of a single clone) in both H. parainfluenzae and H. influenzae Rd. The revertants had lost this small plasmid. Mitomycin C exhibited a plasmid 'curing' effect with a frequency of 'curing' of between 1 and 6% of the surviving clones. It was concluded that the genes conferring NAD independence were located on the small 5.25 kb plasmid.
Insights
Genes for nicotinamide adenine dinucleotide (NAD) independence in Haemophilus parainfluenzae were located on a small plasmid. This plasmid was transferable, and its loss resulted in a return to NAD dependence.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Certain clinical isolates of Haemophilus parainfluenzae exhibit nicotinamide adenine dinucleotide (NAD) independence.
- The genetic basis for this NAD independence in H. parainfluenzae was previously uncharacterized.
Purpose of the Study:
- To determine the location of genes responsible for NAD independence in clinical isolates of Haemophilus parainfluenzae.
- To investigate the role of plasmids in conferring NAD independence.
Main Methods:
- Genes for NAD independence were transferred from H. parainfluenzae to plasmid-free Haemophilus influenzae Rd via transformation.
- Transformants were analyzed for plasmid presence using agarose gel electrophoresis.
- Plasmid curing was induced using Mitomycin C.
Main Results:
- All NAD-independent transformants harbored a single, identical plasmid species of 5.25 kb.
- Spontaneous reversion to NAD dependence occurred at a low frequency, with revertants losing the small plasmid.
- Mitomycin C effectively 'cured' the plasmid in a significant percentage of surviving clones.
Conclusions:
- The genes conferring NAD independence in these Haemophilus parainfluenzae isolates are located on a small 5.25 kb plasmid.
- This plasmid plays a crucial role in the NAD independence phenotype.