DprA/Smf protein localizes at the DNA uptake machinery in competent Bacillus subtilis cells.
BACKGROUND
DprA is a widely conserved bacterial protein and has been shown to confer an important function during transformation in competent cells, possibly through protection of incoming DNA. B. subtilis DprA (called Smf) and has been shown to play an important role during transformation with chromosomal DNA, but its mode of action is unknown.


RESULTS
We show that B. subtilis DprA/Smf is more important for transformation with plasmid DNA than with chromosomal DNA. A functional Smf-YFP fusion localized as discrete foci to the cell pole in a subset of cells grown to competence, dependent on the ComK master transcription factor. Smf-YFP foci colocalized with ComGA-CFP. However, a considerable number of cells having high ComK activity contained Smf dispersed throughout the cytosol and lacked a polar Smf assembly. The absence of polar Smf-YFP foci in these cells strongly correlated with the absence of ComGA-CFP foci, and comGA mutant cells mostly lacked polar Smf-YFP foci. Smf formed polar assemblies in the absence of RecA, and RecA formed dynamic threads after addition of DNA in a smf deletion strain. Upon addition of DNA, Smf-YFP foci relocalized from the poles to the cell centre, dependent on the presence of RecA protein.


CONCLUSIONS
Our data show that Smf is recruited to the polar competence machinery, and that polar Smf assembly requires a functional DNA uptake complex. High ComK levels drive expression of Smf in 20% of all cells grown to competence, but not all competent cells contain a polar DNA uptake machinery, showing that ComK activity is necessary but not sufficient to achieve assembly of the uptake machinery in all cells. Smf and RecA localize independently of each other, in agreement with our finding that Smf is much more important for plasmid transformation than RecA, but RecA influences the dynamic localization pattern of Smf. Our data show that DprA/Smf acts downstream of the DNA uptake machinery, and support the idea that Smf protects incoming ssDNA, possibly in conjunction with RecA.
Tadesse
Serkalem
S
Institut für Mikrobiologie, Faculty for Biology, Schänzlestr, 1, Albert-Ludwigs Universität Freiburg, 79104 Freiburg, Germany. serkalem.tadesse@biologie.uni-freiburg.de
Graumann
Peter L
PL
D019677:Artificial Gene Fusion; D001412:Bacillus subtilis / Q000737:chemistry* / Q000502:physiology*; D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics / Q000502:physiology; D002462:Cell Membrane / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics; D017353:Gene Deletion; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D008565:Membrane Proteins / Q000032:analysis*; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000378:metabolism*; D011938:Rec A Recombinases / Q000032:analysis / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology; D014169:Transformation, Bacterial / Q000502:physiology*
D001426:Bacterial Proteins; C061262:ComA protein, Bacteria; C485182:Cyan Fluorescent Protein; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C093815:DprA protein, bacteria; D008565:Membrane Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; C087449:comK protein, Bacillus subtilis; D011938:Rec A Recombinases