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Novobiocin resistance marker in Haemophilus influenzae that is not expressed on a plasmid
Abstract:
The plasmid pNov2, carrying a cloned chromosomal marker conferring resistance to at least 2.5 micrograms of novobiocin per ml, was constructed with a new Haemophilus influenzae cloning vehicle, pDM2. The novobiocin marker of pNov2 was not normally expressed, but in Rec+ cells approximately one in 10(4) cells in a culture of a transformant became novobiocin resistant, a frequency about four orders of magnitude higher than the spontaneous mutation frequency. Variants of such cells that had lost the plasmid were also novobiocin resistant. Since Rec- cultures bearing pNov2 showed novobiocin resistance only at the normal mutation frequency, we concluded that the Rec+ novobiocin-resistant transformants arose because of a rare recombination between plasmid and chromosome in which the chromosome acquired the novobiocin marker from the plasmid. Evidence is presented that novobiocin sensitivity is dominant over this particular novobiocin resistance marker.
Insights
A novel plasmid, pNov2, facilitated rare recombination events in Haemophilus influenzae Rec+ cells, leading to chromosomal novobiocin resistance. This discovery offers insights into plasmid-chromosome interactions and genetic marker transfer.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Plasmid vectors are crucial tools for genetic manipulation in bacteria.
- Understanding recombination mechanisms is key to bacterial genetics and evolution.
- Novobiocin resistance can serve as a selectable marker in bacterial studies.
Purpose of the Study:
- To construct and characterize a new cloning vector, pNov2, in Haemophilus influenzae.
- To investigate the expression and transfer of a novobiocin resistance marker from a plasmid to the chromosome.
- To elucidate the role of Rec+ (recombination-proficient) cells in this process.
Main Methods:
- Construction of plasmid pNov2 using the pDM2 cloning vehicle.
- Transformation of Haemophilus influenzae strains (Rec+ and Rec-).
- Selection of novobiocin-resistant transformants.
- Analysis of plasmid-chromosome interactions and recombination frequencies.
- Phenotypic analysis of novobiocin sensitivity/resistance.
Main Results:
- Plasmid pNov2 successfully introduced a novobiocin resistance marker into Haemophilus influenzae.
- Rec+ transformants exhibited a significantly elevated frequency (approx. 10^-4) of novobiocin resistance compared to spontaneous mutation rates.
- Novobiocin resistance was observed even in Rec- cells that had lost the plasmid, indicating chromosomal integration.
- Recombination between pNov2 and the H. influenzae chromosome was identified as the mechanism for resistance acquisition.
- Novobiocin sensitivity was found to be dominant over the plasmid-borne resistance marker.
Conclusions:
- The study demonstrates a rare but significant plasmid-mediated recombination event leading to chromosomal novobiocin resistance in Rec+ Haemophilus influenzae.
- This highlights the potential for genetic marker transfer from plasmids to chromosomes via recombination.
- The findings provide a new model for studying plasmid-chromosome interactions and the utility of novobiocin resistance as a selectable marker.