ComEA, a Bacillus subtilis integral membrane protein required for genetic transformation, is needed for both DNA binding and transport.

The competence-related phenotypes of mutations in each of the four open reading frames associated with the comE locus of Bacillus subtilis are described. comEA and comEC are required for transformability, whereas the products of comEB and of the overlapping comER, which is transcribed in the reverse direction, are dispensable. Loss of the comEA product decreases the binding of DNA to the competent cell surface and the internalization of DNA, in addition to exhibiting a profound effect on transformability. The comEC product is required for internalization but is dispensable for DNA binding. ComEA is shown to be an integral membrane protein, as predicted from hydropathy analysis, with its C-terminal domain outside the cytoplasmic membrane. This C-terminal domain possesses a sequence with similarity to those of several proteins known to be involved in nucleic acid transactions including UvrC and a human protein that binds to the replication origin of the Epstein-Barr virus.     

Structural features of DNA in competent Bacillus subtilis

Summary For efficient transformation with B. subtilis, recipient cells must be grown to the state termed competence. Previous findings indicated that such competent cells contained DNA which exhibited about 5% single-strandedness. In this work, the physico-chemical properties of this DNA are compared to artificially nicked DNA. Evidence is presented that breakdown of the host DNA occurs during growth to competence. Inhibition of this breakdown also prevents the formation of partially single-stranded chromosomes within the competent cells. Use of this DNA as donor in transformation studies indicated a deficiency in biological activity within specific genes. Of three models considered, it is concluded that the results are best explained by the occurrence of single-stranded gaps within the chromosomes of competent cells.     

NucA is required for DNA cleavage during transformation of Bacillus subtilis

We have re-examined the roles of nucA and nin, in the transformation of Bacillus subtilis as conflicting accounts have been presented concerning the importance of these genes for transformation. The present report demonstrates that nucA deficiency lowers the rate of DNA transport and that NucA is needed for the double-strand cleavage of transforming DNA, probably acting directly as an endonuclease. A relative paucity of DNA termini, resulting from the absence of this endonuclease activity, most probably accounts for the decreased transport rate. NucA is a bitopic integral membrane protein, with its C-terminus external to the membrane where it is appropriately located to effect the cleavage of bound transforming DNA. We have also investigated the roles of the known competence genes in the DNA processing that accompanies transformation in B. subtilis. The genes that are required for DNA transport (comEA, comEC and comFA) are also required for the degradation of the non-transforming strand that accompanies internalization, but comEC and comFA are not needed for the double-strand cleavage that occurs external to the cell membrane.     

Interspecific transformation of Bacillus subtilis competent cells by chromosomal DNA in lysates of protoplasts of Bacillus amyloliquefaciens

Competent cells of Bacillus subtilis were transformed with chromosomal DNA in lysates of protoplasts of B. subtilis or B. amyloliquefaciens. The interspecific transformation frequency of B. subtilis by cysA in a conserved region was 3.1 x 10(4) transformants per microg DNA, 60 times higher than that for conventional transformation using purified DNA. Increased interspecific transformation frequencies of B. subtilis were also observed for arg-1, lys-1, leuB, aroG, thr-5, hisH, or metC markers outside the conserved region (3.1 x 10 approximately 5.2 x 10(2) transformants per microg DNA). An interspecific cotransformation ratio (33-50%) as high as an intraspecific one (46%) using purified DNA was also detected between cysA and rpsL markers, which are separated by 16 kb on the B. subtilis chromosome. Interspecific double transformation of the cysA-arg-1 or cysA-metC marker was observed, which have not been detected for conventional transformation. The involvement of mutS in the interspecific transformation was not significant.     

DNA synthesis in competent Bacillus subtilis cells.

Competent cells of Bacillus subtilis incorporate degradation products from transfecting DNA into their chromosomal DNA. The sensitivity of this incorporation to inhibitors of bacterial DNA synthesis [phage infection or 6-(p-hydroxyphenylazo)-uracil] suggests that semiconservative DNA synthesis can occur in competent cells.     

DNA repair in competent cells of Bacillus subtilis.

A series of isogenic transformable strains of Bacillus subtilis carrying the uvr-19 or rec-43 mutation or both were constructed. Both mutations made competent cells defective in repairing UV-irradiated cellular or transforming DNA, and their effects were additive in a doubly deficient strain, suggesting that two repair processes, requiring uvr-19+ and rec-43+ gene products, are independently functional in competent cells of B. subtilis.     

Intramolecular recombination during plasmid transformation of Bacillus subtilis competent cells.

We have constructed plasmids carrying direct internal repeats 260-2000 bp long. Monomers of such plasmids transformed Bacillus subtilis competent cells. The efficiency of transformation varied with the square of the length of repeats. The transformed clones harbored either the entire transforming plasmid and the plasmid arising by recombination between the repeats, or only the latter plasmid. Internally-repeated plasmids linearized by in vitro cleavage with restriction endonuclease could transform, yielding clones which exclusively harbored a plasmid resulting from recombination between the repeats. When the transforming plasmid carried repeats which differed slightly, conversion of one repeat into the other could occur. The following model of plasmid transformation accounts for these data: (1) plasmid DNA is cleaved and rendered linear in contact with competent cells; (2) a linear, at least partially double-stranded plasmid molecule is introduced or formed by repair within the cell; (3) a circular viable plasmid is produced by recombination between repeats carried on this molecule; (4) alternatively, a viable plasmid is produced by repairing the cut within one of the repeats by DNA synthesis which uses the other repeat as a template.     

Direction of DNA entry in competent cells of Bacillus subtilis

Direction of DNA entry in Bacillus subtilis competent cells was studied using molecules in which only one of the two strands was radioactively labelled. The label was either distributed homogeneously or was localized in a small region of the strand, in the centre or at one of the ends. Regardless of the distribution and the position of the label, similar amounts of radioactivity were taken up by the cells exposed to the labelled molecules. This suggests that DNA enters B. subtilis either by two different uptake systems having opposite polarities, or by a single non-polar system.     

The initial attachment of transforming DNA to competent Bacillus subtilis.

Summary The initial attachment of transforming DNA to competent Bacillus subtilis is temperature independent between 25° and 45°. However, below 15° there is a significant reduction in the amount of DNA attached to competent cells. The DNA that is attached at 4° can lead to transformation or interfere effectively with the subsequent attachment of a distinctive DNA when the cells are shifted to a permissive temperature (37°). These data suggest that the attachment of DNA at 4° is to sites normally involved in the transformation process. The amount of DNA that is initially attached to the bacteria at 4° or 37° after perturbation of the cells by ionic strength changes, repetitive washings, or periodate oxidation varies with the temperature at which the treatment occurs. These results are consistent with a reorientation of the DNA attachment sites upon lowering the temperature to 4°, such that their affinity for DNA and susceptibility inhibitory treatments are reduced.National Institutes of Health Research Career Program Awardee, CA-K3-6487 during a portion of this investigation.     

Features of transformation of competent cells and Bacillus subtilis protoplasts by integrative vectors

The effect of structural peculiarities of DNAs from integrative plasmids on the transformation activity was studied. Monomeric forms of the plasmids can only transform B. subtilis competent cells, when plasmid selective marker is inserted into chromosomal fragment within the plasmid. Polymeric forms are needed for efficient transformation. Both single- and double-stranded DNAs of integrative plasmids transform no B.subtilis protoplasts, this being irrespective of plasmid structure.     

The three-layered DNA uptake machinery at the cell pole in competent Bacillus subtilis cells is a stable complex

Many bacteria possess the ability to actively take up DNA from the environment and incorporate it into the chromosome. RecA protein is the key protein achieving homologous recombination. Several of the proteins involved in the transport of DNA across the cell envelope assemble at a single or both cell poles in competent Bacillus subtilis cells. We show that the presumed structure that transports DNA across the cell wall, the pseudopilus, also assembles at a single or both cell poles, while the membrane receptor, ComEA, forms a mobile layer throughout the cell membrane. All other known Com proteins, including the membrane permease, localize again to the cell pole, revealing that the uptake machinery has three distinct layers. In cells having two uptake machineries, one complex is occasionally mobile, with pairs of proteins moving together, suggesting that a complete complex may lose anchoring and become mobile. Overall, the cell pole provides stable anchoring. Only one of two uptake machineries assembles RecA protein, suggesting that only one is competent for DNA transfer. FRAP (fluorescence recovery after photobleaching) analyses show that in contrast to known multiprotein complexes, the DNA uptake machinery forms a highly stable complex, showing little or no exchange with unbound molecules. When cells are converted into round spheroplasts, the structure persists, revealing that the assembly is highly stable and does not require the cell pole for its maintenance. High stability may be important to fulfill the mechanical function in pulling DNA across two cell layers.     

A membrane protein with similarity to N-methylphenylalanine pilins is essential for DNA binding by competent Bacillus subtilis.

In a cloned copy of comG open reading frame 3 (ORF3), an in-frame deletion was generated by site-directed in vitro mutagenesis, removing the coding sequence for 15 amino acids from the central portion of this pilin-related protein. The mutagenized ORF3 was incorporated into the Bacillus subtilis chromosome, replacing the wild-type ORF3. The presence of the deleted ORF3 in the chromosome, as confirmed by Southern analysis, was associated with the complete loss of competence by the mutant strain. The ability of the mutant cells to bind exogenous radiolabeled DNA was reduced to the level of nonspecific binding of DNA by noncompetent cells. The chromosomal ORF3 mutation was partially complemented in trans by a plasmid-encoded wild-type ORF3 copy under PSPAC control upon induction of the PSPAC promoter. Using antiserum raised against a synthetic 14-mer oligopeptide deduced from the ORF3 sequence, an immunoreactive band of approximately the expected molecular size was obtained in Western blot (immunoblot) experiments with extracts of cells containing the plasmid-encoded inducible gene. A signal was also detected when cells harboring the chromosomal wild-type or mutant ORF3 in single copy were grown in competence medium. This signal was detected only in the light-buoyant-density (competent) cell fraction and only after the transition from the exponential to the stationary growth phase. In cell fractionation experiments with competent cell extracts, the immunoreactive protein was found in both the NaOH-insoluble and -soluble membrane fractions and was sensitive to proteinase K treatment of either protoplasts or whole cells.     

Formation of competent Bacillus subtilis cells.

The process of competent cell formation for transformation has been studied with early-stationary-phase (T1) cells of Bacillus subtilis which had been grown in an enriched Spizizen minimal medium and transferred to a second synthetic medium. Rifampin, chloramphenicol, and tunicamycin were strong inhibitors of competent cell formation, as well as vegetative growth. After formation, competent cells were no longer sensitive to the above agents. Methicillin and an inhibitor of chromosomal replication, hydroxyphenylazouracil, did not inhibit the development of competence. A D-alanine-requiring mutant strain developed competence even in the absence of D-alanine in the second medium. A T1-stage culture showed the activity of extracellular serine protease which is necessary for sporulation. Competent cell formation was completely blocked by 0.7 M ethanol, which is a specific inhibitor of early events during sporulation, including forespore septum formation. Competent cells were formed even in media which supported sporulation. The development of competence was also studied with spo0 mutants at 10 different loci. Most spo0 mutations repressed the development of competence except for spo0C, spo0G, and spo0J. These results suggest that competent cells are formed from early sporulating cells with the synthesis of cell wall materials and by factors whose genes are activated by the supply of nutrients. It is suggested that common steps are involved both in forespore septation and in competent cell formation.     

Chromosomal transformation in Bacillus subtilis is a non-polar recombination reaction

Natural chromosomal transformation is one of the primary driving forces of bacterial evolution. This reaction involves the recombination of the internalized linear single-stranded (ss) DNA with the homologous resident duplex via RecA-mediated integration in concert with SsbA and DprA or RecO. We show that sequence divergence prevents Bacillus subtilis chromosomal transformation in a log-linear fashion, but it exerts a minor effect when the divergence is localized at a discrete end. In the nucleotide bound form, RecA shows no apparent preference to initiate recombination at the 3′- or 5′-complementary end of the linear duplex with circular ssDNA, but nucleotide hydrolysis is required when heterology is present at both ends. RecA·dATP initiates pairing of the linear 5′ and 3′ complementary ends, but only initiation at the 5′-end remains stably paired in the absence of SsbA. Our results suggest that during gene transfer RecA·ATP, in concert with SsbA and DprA or RecO, shows a moderate preference for the 3′-end of the duplex. We show that RecA-mediated recombination initiated at the 3′- or 5′-complementary end might have significant implication on the ecological diversification of bacterial species with natural transformation.