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Possible mechanism for donor DNA binding and transport in Haemophilus
Abstract:
Morphological differences were observed in competent and noncompetent Haemophilus parainfluenzae and Haemophilus influenzae when thin sections of these cells were examined by electron microscopy. The membranous extensions present on the surface of competent H. parainfluenzae cells disappeared on treatment with transforming DNA, while vacuole-like structures appeared in the periplasm. Noncompetent cells had 1/5th as many extensions on their surface as competent cells, and no vacuoles were observed after treatment with homologous DNA. Competent cells treated with radiolabeled DNA were disrupted and the clarified lysate was centrifuged on CsCl density gradients. Material having a density of 1.34 g/ml was found to contain the majority of the DNase-resistant radioactive DNA recovered from the bacteria and was shown by electron microscopy to be composed of membrane vesicles. The polypeptide composition of this dense membrane fraction was similar to that of H. parainfluenzae outer membrane.
Insights
Competent Haemophilus parainfluenzae cells exhibit surface membrane changes upon DNA uptake, forming periplasmic vacuoles. These vacuoles, containing DNA, originate from the outer membrane, suggesting a role in genetic transformation.
Area of Science:
- Microbiology
- Molecular Biology
- Cell Biology
Background:
- Haemophilus parainfluenzae and Haemophilus influenzae are bacteria known to undergo natural genetic transformation.
- Competence, the ability to uptake exogenous DNA, involves complex cellular mechanisms.
- Understanding the morphological changes associated with competence is crucial for elucidating transformation pathways.
Purpose of the Study:
- To investigate the morphological alterations in competent versus noncompetent Haemophilus cells during DNA interaction.
- To characterize the structures involved in DNA binding and uptake in competent H. parainfluenzae.
Main Methods:
- Electron microscopy of thin-sectioned competent and noncompetent Haemophilus parainfluenzae and H. influenzae.
- Treatment of competent cells with transforming DNA and observation of morphological changes.
- Disruption of competent cells treated with radiolabeled DNA, followed by ultracentrifugation on CsCl density gradients.
- Electron microscopic and biochemical analysis of isolated cellular fractions.
Main Results:
- Competent H. parainfluenzae displayed surface membranous extensions that disappeared upon treatment with transforming DNA.
- Vacuole-like structures were observed in the periplasm of competent cells after DNA treatment.
- Noncompetent cells showed significantly fewer surface extensions and no vacuole formation.
- A dense membrane fraction (1.34 g/ml) containing DNase-resistant DNA was isolated, composed of membrane vesicles resembling the outer membrane.
Conclusions:
- The study reveals distinct morphological differences between competent and noncompetent Haemophilus cells.
- Membrane vesicles derived from the outer membrane appear to be involved in the uptake and processing of transforming DNA.
- These findings provide insights into the physical mechanisms of DNA binding and internalization during bacterial transformation.