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Uptake of transforming DNA in Gram-positive bacteria: a view from Streptococcus pneumoniae
Mathieu Bergé1, Miriam Moscoso, Marc Prudhomme
1Laboratoire de Microbiologie et Génétique Moléculaire, UMR 5100 CNRS-Université Paul, Sabatier, 118 route de Narbonne, 31062 Toulouse Cedex, France.
Abstract:
In a working model for the uptake of transforming DNA based on evidence taken from both Bacillus subtilis and Streptococcus pneumoniae, the ComG proteins are proposed to form a structure that provides access for DNA to the ComEA receptor through the peptidoglycan. DNA would then be delivered to the ComEC-ComFA transport complex. A DNA strand would be degraded by a nuclease, while its complement is pulled into the cell by ComFA through an aqueous pore formed by ComEC. The nuclease is known in S. pneumoniae only as EndA. We have examined the processing (i.e. binding, degradation and internalization) of DNA in S. pneumoniae strains lacking candidate uptake proteins. Mutants were generated by transposon insertion in endA, comEA/C, comFA/C, comGA and dprA. Processing of DNA was abolished only in a comGA mutant. As significant binding was measured in comEA mutants, we suggest the existence of two stages in binding: surface attachment (abolished in a comGA mutant) required for and preceding deep binding (by ComEA). Abolition of degradation in comGA and comEA mutants indicated that, despite its membrane location, EndA cannot access donor DNA by itself. We propose that ComEA is required to deliver DNA to EndA. DNA was still bound and degraded in comEC and comFA mutants. We conclude that recruitment of EndA can occur in the absence of ComEC or ComFA and that EndA is active even when the single strands it produces are not pulled into the cell. Finally, inactivation of dprA had no effect on the internalization of DNA, indicating that DprA is required at a later stage in transformation.
Insights
DNA uptake in Streptococcus pneumoniae requires the ComGA protein for initial DNA binding and subsequent degradation by the EndA nuclease. ComEA facilitates DNA delivery to EndA, while ComEC and ComFA are not essential for initial processing.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Bacterial transformation involves DNA uptake through the cell envelope.
- The ComG proteins, ComEA, ComEC, and ComFA are implicated in DNA uptake in Bacillus subtilis and Streptococcus pneumoniae.
- EndA is a nuclease in S. pneumoniae involved in DNA processing.
Purpose of the Study:
- To investigate the roles of candidate DNA uptake proteins in Streptococcus pneumoniae.
- To elucidate the sequential steps of DNA binding, degradation, and internalization during transformation.
- To determine the specific functions of ComGA, ComEA, EndA, ComEC, ComFA, and DprA in DNA processing.
Main Methods:
- Generation of Streptococcus pneumoniae mutants using transposon insertion in genes encoding candidate uptake proteins (endA, comEA/C, comFA/C, comGA, dprA).
- Analysis of DNA processing, including binding, degradation, and internalization, in wild-type and mutant strains.
- Comparative analysis of DNA processing efficiency across different mutant strains.
Main Results:
- DNA processing was abolished only in the comGA mutant, indicating ComGA's essential role.
- ComEA is involved in deep DNA binding, with ComGA required for initial surface attachment.
- EndA-mediated DNA degradation requires ComEA for access to donor DNA.
- DNA binding and degradation occurred in comEC and comFA mutants, suggesting EndA can function independently of ComEC/ComFA.
- DprA inactivation did not affect DNA internalization, suggesting its role in a later stage of transformation.
Conclusions:
- ComGA is essential for the initial attachment and subsequent processing of transforming DNA.
- ComEA acts as a crucial mediator, delivering DNA to the EndA nuclease.
- EndA can degrade DNA independently of the ComEC-ComFA transport complex.
- DprA plays a role in a later stage of DNA transformation, after initial processing.