DprA/Smf protein localizes at the DNA uptake machinery in competent Bacillus subtilis cells
© Tadesse and Graumann; licensee BioMed Central Ltd. 2007
Received: 29 March 2007
Accepted: 28 November 2007
Published: 28 November 2007
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.
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.
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.
Bacterial competence describes the physiological state that permits the uptake of exogenous DNA in macromolecular form for integration into the chromosome via homologous recombination [1, 2]. Competence has been found in a range of bacterial phyla, including the Gram positive Bacillus subtilis. Because many pathogenic bacteria have the ability to gain competence, this process is important in light of spreading of antibiotic resistance genes. B. subtilis cells develop competence when they enter the stationary phase of growth in response to nutrient limitation [1, 2]. During this developmental process, only a fraction of the cells transiently differentiate into competent cells that have physiological characteristics different from those of non-competent cells. This phenomenon is called bistability, in which cells of a single population switch reversibly between two transcriptional states [3–5]. In B. subtilis, 10–20% of the cells in a given population differentiate to the competence state, which lasts for 3–5 hours, after which cells turn of transcription of competence genes and exit the so called K-state [2, 6].
The master competence transcription factor, ComK, regulates at least 165 genes during competence [6–8], and has been shown to become active exclusively in only those few competent cells (10–20%) [9, 10]. At least 16 competence proteins are needed to bind environmental DNA to the cell membrane and transport this DNA into the cytosol, where it is available for recombination with resident DNA . Within the competence machinery, proteins generally work at three different levels. The process of binding DNA at the surface of the cell requires a pseudopilus-like structure (possibly guiding DNA through the peptidoglycan layer) and a DNA-binding protein (ComEA) , and is reversible . Transport of DNA is mediated through a channel in the cell membrane (ComEC) , and is highly powerful and processive . DNA transport depends on the proton motive force , and may be driven by an additional energy source. The transport subdivision includes ATPases, like ComGA  or ComFA [16, 17], which may enable coupling of ATP hydrolysis and transport. The third group of proteins in the machinery are cytosolic proteins such as RecA, and RecN, that interact with ComGA  and mediate homologous recombination, and proteins of unknown function such as YwpH/SsbB, which is homologous to ssDNA-binding SSB (SsbA) protein .
Interestingly, the competence machinery assembles at a single cell pole in competent B. subtilis cells. This includes the DNA uptake machinery  and three cytosolic proteins, RecA, RecN and YwpH [17, 18]. Upon addition of DNA to the cells, RecA protein forms dynamic filamentous structures, termed threads, which are thought to guide ssDNA that is taken up through the ComEC channel to the nucleoid. Here, RecA mediates strand exchange between the chromosomal DNA duplex and taken up DNA, given that extensive homology exists between ssDNA and the chromosome . Thus, competence in B. subtilis is a temporally and spatially highly organized process.
DprA (D NA pr ocessing protein) is a widely conserved protein involved in competence in Hemophilus infuenzae, Helicobacter pylori and Campylobacter jejuni, and is called Smf in B. subtilis or CilB or Dal in Streptococcus pneumoniae. DprA orthologs have been shown to impart an important role in transformation with chromosomal DNA or with plasmid DNA [6, 7, 19–21], and the S. pneumoniae ortholog is thought to protect incoming DNA during transformation , but genetic and biochemical functions of DprA/Smf are not yet understood. Here we investigate B. subtilis Smf using genetic, cell biological and biochemical tools. B. subtilis DprA/Smf is important for transformation with chromosomal DNA, but even more so with plasmid DNA. DprA/Smf shows localization characteristics similar to but also distinct from representatives of the two groups of proteins of the competence machinery that play a major role in DNA uptake (ComGA) and DNA transformation (RecA and RecN), suggesting that DprA/Smf plays an important role at the interface between the uptake and the recombination machineries.
DprA/Smf plays an important role in transformation of chromosomal DNA and of plasmid DNA
Transformation efficiency of mutant cells compared to wild type cells
E. coli plasmid (integrative)
Plasmid (self replicating)
79.5 (± 4.5)
102 (± 5)
96.8 (± 3.5)
10.8 (± 2.0)
0.58 (± 0.25)
1.69 (± 0.25)
0.11 (± 0.05)
0.49 (± 0.05)
49.5 (± 2.5)
0.08 (± 0.04)
0.24 (± 0.06)
0.94 (± 0.45)
Smf does not influence the localization of ComGA, or RecA or RecN dynamics
To find out if Smf affects the function of RecA during transformation in competent cells, we monitored the localization of GFP-RecA in smf mutant cells grown to competence. Similarly to wild type cells, GFP-RecA localized to the cell poles in 20% of the cells in the absence of Smf (Figure 1B, compare with 1A), and formed dynamic threads after addition of DNA, irrespective of the presence or absence of Smf (Figure 1D, compare with 1C). Thus, the two important forms of RecA – accumulation at the competence cell pole and formation of threads – operate apparently normally after loss of Smf protein. Similarly, RecN-YFP localized to a single cell pole in smf mutant cells (data not shown). These experiments show that Smf does not act directly upstream of RecA or of RecN, and support the idea that Smf and RecA perform somewhat distinct functions with regard to transformation. Because formation of RecA threads depends on DNA uptake, the results suggest that Smf acts after DNA uptake, but not downstream of RecA during homologous recombination.
Smf localizes throughout the cytosol in competent cells, but also as discrete foci at a single cell pole in most but not all competent cells
Smf colocalizes with ComGA at the cell pole, independent of RecA, and localizes throughout competent cells lacking polar ComGA protein
To find out why Smf has two distinct patterns of localization, we combined the Smf-YFP fusion with a ComGA-CFP fusion. ComGA-CFP localized to a single cell pole in competent cells in 13% of all cells grown to competence, as previously reported (Figure 2F). In 97% of these cells (i.e. 12.5% of all cells), polar ComGA-CFP foci colocalized with Smf-YFP foci, while 3% of these cells showed cytosolic localization of Smf-YFP and polar ComGA-CFP localization (Figure 2F, > 350 cells analysed). Intriguingly, an additional 9% of all cells grown to competence contained high Smf-YFP fluorescence throughout the cells, but these did not contain ComGA-CFP foci (Figure 2F). Therefore, Smf-YFP foci strongly correlate with the presence of ComGA-CFP foci, and thus with the presence of the competence machinery, while in the absence of the polar complex, Smf-YFP is dispersed throughout the cell. To obtain further information, we visualized Smf-YFP in competent comGA mutant cells. Smf-YFP foci were present in only 0.7% of the mutant cells grown to competence, whereas 17% of the cells had high Smf-YFP levels throughout the cells (with 350 cells analysed; in about 1/3 of these cells, Smf frequently localized in patches or foci, rather than uniformly throughout the cytosol) (Figure 2G). Thus, the presence of ComGA is important, but not required, for the formation of polar Smf-YFP foci. We also monitored the localization of Smf-YFP in cells lacking the ComEC DNA uptake channel. Only 2% of comEC mutant cells contained polar Smf-YFP foci, while 18% of all cells grown to competence contained solely cytosolic Smf (340 cells analysed) (Figure 2H). Therefore, the presence of the competence machinery rather than specifically ComGA or ComEC is required for efficient recruitment of Smf to the cell pole. It is possible that another protein of the competence complex serves as a specific binding partner for Smf. In any event, our results also show that several competent cells (that is cells with sufficient ComK activity to drive expression of Smf) do not contain a fully assembled polar competence machinery, showing that high ComK activity is not sufficient to grant the assembly of the polar DNA uptake machinery.
To further investigate determinants of polar localization of Smf, we moved the Smf-YFP fusion into recA or recN mutant cells. Smf-YFP foci were still present in cells lacking RecA (Figure 2I) or RecN (data not shown), showing that recruitment of Smf to the pole is independent of the presence of the two homologous recombination proteins. These data support the findings that RecA and Smf localize independently of each other.
Smf changes its pattern of localization in response to addition of DNA, dependent on RecA
Intriguingly, Smf-YFP did not alter its polar localization pattern after addition of chromosomal DNA in the absence of RecA; between 12 and 13% of recA mutant cells grown to competence showed polar Smf-YFP foci between 10 and 60 min after addition of DNA (Figure 3G, 260 cells analysed). Thus, although Smf and RecA localize to the cell pole independently of each other, and RecA forms threads independently of Smf, dynamic relocalization of Smf depends on RecA, showing that a connection exists between the two proteins.
Our work provides important novel insight into the function of DprA (also called Smf or CilB), a protein widely conserved in bacteria, some of which possess natural competence. In competent S. pneumoniae cells, the half-life of incoming DNA was strongly reduced in the absence of DprA, and also in the absence of RecA, suggesting that DprA may physically protect incoming DNA before loading of RecA occurs . DprA has been shown to play an important role in the transformation with chromosomal DNA in B. subtilis [6, 7, 19–21]. We have found that transformation with plasmid DNA is even more severely reduced in smf (dprA) mutant cells compared to transformation with chromosomal DNA. Transformation of plasmids requires the full establishment of the plasmid from single stranded DNA fragments that are taken up by the DNA uptake machinery, and does not involve homologous recombination (HR) with the chromosome, while single stranded chromosomal DNA fragments can be directly used for HR without the need to generate dsDNA intermediates. Our genetic experiments show that DprA/Smf is not as important for transformation with chromosomal DNA as RecA, but more important for transformation with a self replicating plasmid than RecA, suggesting that DprA/Smf and RecA have somewhat distinct functions during transformation. However, these data do not rule out that both proteins act in conjunction. How might DprA confer its important function during competence? Several lines of evidence suggest that DprA/Smf acts at a step directly following DNA uptake. To test if DprA/Smf functions downstream or upstream of RecA, or in conjunction with RecA, we monitored the localization of RecA in the absence of Smf. In competent B. subtilis cells, RecA localizes to a single cell pole that contains the DNA uptake machinery . Upon addition of DNA, RecA forms dynamic filamentous structures termed threads that extend from the DNA uptake machinery towards and onto the nucleoids in the cell centre. A RecA allele that is unable to form threads after addition of DNA is non functional, indicating that these structures are active intermediates during transformation. The absence of DprA/Smf did not alter the pattern of localization of RecA, the protein localized to a single pole in competent cells and formed threads indistinguishable from those in wild type cells after addition of DNA. Similarly, Smf did not affect the specific localization of RecN, another proteins involved in HR. Thus, Smf does not appear to act upstream of RecA, but possibly in parallel with RecA. This idea is supported by cytological data gained from the investigation of a fully functional Smf-YFP fusion. Smf-YFP was present throughout the cytosol in about 20% of cells grown to competence. In agreement with DNA array data [6–8], the expression of DprA/Smf was strictly dependent on ComK. However, about 10–12% of the cells containing cytosolic Smf-YFP contained Smf-YFP foci at a single cell pole, which colocalized with ComGA, and thus with the DNA uptake machinery . Thus, DprA/Smf displays two different patterns of localization in competent cells, absence or presence of polar assemblies in parallel with diffusely localized protein. DprA/Smf assembly at the cell pole was strongly reduced in the absence of ComGA or of ComEC. Conversely, Smf-YFP still formed polar foci in cells lacking RecA, showing that Smf and RecA localize independently, but in association with the DNA uptake machinery. Intriguingly, addition of DNA to competent cells strongly influenced the localization of Smf-YFP. Already 10 min after addition of DNA, the number of polar Smf-YFP foci decreased, frequently (but not always) concomitant with the appearance of one or several cytosolic Smf-YFP foci. Between 20 and 30 min after addition of DNA, these assemblies largely disappeared, and Smf became dispersed throughout the cytosol. These data show that Smf is not statically associated with the polar uptake machinery, but can transiently dissociate from the pole. Indeed, we found that purified Smf is a soluble protein, and forms a monomer in solution (data not shown). Interestingly, dynamic relocalization of Smf was dependent on RecA, because Smf remained at the cell pole after addition of DNA in recA mutant cells. These data are compatible with and support the idea that DprA/Smf protects incoming ssDNA from nucleolytic attack , which will be transported onto the nucleoids for homologous recombination. This function will be important during transformation with chromosomal DNA that can be directly used for integration into the chromosome. Stabilization of incoming ssDNA is probably even more important for establishment of a dsDNA plasmid (requiring ssDNA strand annealing and DNA synthesis), which would explain why DprA/Smf is even more important during plasmid transformation. Our finding that polar Smf foci dissipate upon addition of DNA, dependent on RecA protein, is consistent with our hypothesis that RecA threads are involved in the transport of ssDNA from the pole to the chromosome, and indicates that Smf binds to incoming ssDNA (directly or indirectly) and is transported away from the pole. However, this last idea is still highly speculative. It will be important to find out if DprA/Smf interacts directly with RecA, and to identify other factors that interact with DprA/Smf, to further elucidate its function(s) during transformation.
Our results also provide insight into the assembly of the polar competence machinery. The polar uptake machinery has been shown to transiently assemble and later disassemble at the pole in competent cells, dependent of ComK activity. In our experiments, many cells contained high ComK activity, as well as a high amount of Smf, and thus also of all other competence proteins. However, a considerable proportion of these cells did not contain an assembly of Smf at a cell pole, even after an extended time after the onset of competence, but rather solely Smf located throughout the cytosol. These cells did not contain a fully assembled DNA uptake machinery, as judged from the absence of ComGA in cells lacking a polar Smf assembly. Thus, Smf molecules of the dispersed cytosolic pool relocalize to the cell pole only in cells containing the uptake machinery. These results suggest that high ComK activity is necessary but not sufficient to ensure assembly of the DNA uptake machinery, indicating another level of regulation exists for the assembly of the complete competence machinery. Thus, competence in B. subtilis serves as a model for the investigation of assembly of a large membrane-protein complex, and may yield important new insight into this fundamentally important process.
Bacterial strains, growth conditions and transformation
Escherichia coli XL-1Blue (Stratagene) was used for cloning experiments grown in Luria-Bertani (LB) rich media supplemented with 50 μg/ml ampicillin. Cells were grown to competence as described previously by Dubnau and Davidoff-Abelson , with appropriate antibiotics for selection. Antibiotics were used at the following concentrations: chloramphenicol (Cm) at 5 μg/ml, kanamycin (Kan) at 10 μg/ml, tetracycline (Tet) 10 μg/ml and Erythromycin (Ery) 1 μg/ml.
Construction of vectors and strains
Primers used in this study
smf c-ter up
smf c-ter dwn
Forward smf topo Reverse smf topo
smf-yfp (at original locus; Cmr)
smf-cfp (at original locus; Cmr)
smf-yfp, comK-cfp (Cmr, Kanr)
comK::Ery, smf-yfp (Cmr)
smf-yfp, comGA-cfp (Cmr, Kanr)
comGA::Ery, smf-yfp (Cmr)
comEC::Ery, smf-yfp (Cmr)
recA::Cm, smf-yfp (Tetr)
smf::Ery, gfp-recA (pXyl)
recN::Cm, smf-yfp (Tetr)
Transformation efficiency test
To carry out the plating experiment for transformation efficiency of the different strains, an OD260 measurement was taken to determine the concentration of the chromosomal DNA, self replicating shuttle plasmid (pDG145, kanamycin resistance), and integrating plasmid (pYkoV-YFP, chloramphenicol resistance). The measurement of optical density for competent cells was carried out at an OD600. After addition of DNA for 30 min, cells were serially diluted and plated onto LB agar having the appropriate antibiotics to be incubated at 37°C for 24 hours. Transformation efficiency was determined as ratio of transformants versus viable cells, and numbers for wild type cells from 3 independent experiments were considered as 100%.
Fluorescence microscopy was performed on an Olympus AX70 microscope. The respective competent cells were mounted on agarose gel coated slides. Images were acquired with a digital charge-coupled device camera (Princeton Instruments MicroMax) driven by Metamorph 5.0 program (Universal Imaging Corp., USA). DNA was stained with 4',6-diamidino-2-phenylindole (DAPI; final concentration, 0.2 ng/ml), and membranes were stained with FM-4-64 (final concentration, 1 nM). Filters used were: DAPI – ex360–370, dc400, em420–460, CFP: ex D436/20, dc 455DCLP, em D480/40, YFP: ex HQ500/20, dc Q515LP, em HQ535/30, GFP – ex460–495, dc505, em510–550, FM4–64 ex480–550, dc570, em590. For studies employing addition of DNA to competent cells, 5 μg of chromosomal DNA or 5 μg of plasmid DNA was added to 100 μl of culture containing about 2 × 109 cells, corresponding to amounts of DNA that give a reasonable number of transformants. Chromosomal or plasmid DNA containing various different resistant genes were employed.
We thank Dr. M. Ogura and Dr. T. Tanaka from Tokai University for the kind gift of the smf mutant strain. This work was supported by a grant from the Deutsche Forschungsgemeinschaft.
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