Crystal protein formed by Bacillus subtilis cells.

Electron microscopic investigation of ultrathin sections of Bacillus subtilis Cgr4 cells revealed the presence of crystal-like inclusions which were formed of spheric homogeneous subunits. The frequency of cells with a crystal-like inclusion in the culture approached 1%. The appearance of the crystal protein in cells coincided in time with spore morphogenesis. However, the process of crystal protein formation and sporulation are two alternatives: the cells either form the crystal protein or continue spore morphogenesis. Fractionation of cells in the stationary growth phase on a Percoll density gradient showed that the cells containing the crystal protein accumulated in the fraction corresponding to a 1.14-g/ml Percoll density. The cells were disintegrated by sonication, and alkaline-extracted proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After sodium dodecyl sulfate-gel electrophoresis, the fraction enriched with crystal-containing cells showed practically a single band with a molecular weight of 47,000 that corresponded to the crystal-forming protein. The antigenic features and amino acid composition indicated certain similarities between the crystal-forming protein in B. subtilis Cgr4 cells and the spore coat protein.     

ComC is required for the processing and translocation of ComGC, a pilin-like competence protein of Bacillus subtilis

ComGC is a cell surface-localized protein required for DNA binding during transformation in Bacillus subtilis. It resembles type IV prepilins in its N-terminal domain, particularly in the amino acid sequence surrounding the processing cleavage sites of these proteins. ComC is another protein required for DNA binding, which resembles the processing proteases that cleave type IV prepilins. We show here that ComGC is processed in competent cells and that this processing requires ComC. We also demonstrate that the PilD protein of Neisseria gonorrhoeae, a ComC homologue, can process ComGC in Escherichia coli, and that the ComC protein itself is the only B. subtilis protein needed to accomplish cleavage of ComGC in the latter organism. Based on NaOH-solubility studies, we have shown that in the absence of ComC, but in the presence of all other competence proteins, B. subtilis is incapable of correctly translocating ComGC to the outer face of the cell membrane. Finally, we show that ComGC can be cross-linked to yield a form with higher molecular mass, possibly a dimer, and present evidence suggesting that formation of the higher mass complex takes place in the membrane, prior to translocation. Formation of this complex does not require ComC or any of the comG products, other than ComGC itself.     

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.     

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.     

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.     

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.     

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.     

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.     

ComEA is a DNA receptor for transformation of competent Bacillus subtilis

Competent cells of Bacillus subtilis efficiently bind and internalize DNA. ComEA and the seven proteins encoded by the comG operon are required in vivo for the binding step. We show here that ComEA, a bitopic membrane protein, is itself capable of high-affinity DNA binding. A domain necessary for DNA binding is located at the C-terminus of ComEA. Proteins with similar 60–80 amino acid residue domains are widespread among bacteria and higher organisms. ComEA shows a marked preference for double-stranded DNA and can bind to oligomers as small as 22 bp in length. DNA binding by ComEA exhibits no apparent base sequence specificity. Using a membrane vesicle DNA-binding assay system we show that in the absence of cell wall, ComEA is still required for DNA binding, whereas the requirement for the ComG proteins is bypassed. We conclude that the ComG proteins are needed in vivo to provide access of the binding domain of ComEA to exogenous DNA. Possible specific roles for the ComG proteins are discussed.     

DNA-dependent ATPases in Bacillus subtilis mutants and in competent cells

Three DNA-dependent ATPases (gamma phosphohydrolases) can be isolated from Bacillus subtilis cells. We studied these enzymes in a number of mutants deficient in recombination or repair functions (rec, uvr) and in competent cells. The recA mutant studied had lower ATPase II activity, while competent cells had higher ATPase I activity, in comparison with the parental strain not brought to competence.     

Characteristics of a complex formed by a nonintegrated fraction of transforming DNA and Bacillus subtilis recipient cell constituents.

Summary Nonintegrated transforming DNA was isolated from recipient cell lysates as a complex with cellular constituents (natural complex) and separated from free proteins on CsCl density gradients. Sensitivity of DNA in this complex to digestion with endonuclease S₁, liberation of denatured donor molecules by treatment of the cell lysates with phenol, as well as previously described liberation of single-stranded donor DNA by heating with detergents, pointed to the single-stranded nature of the donor DNA in the complex. About 1% of radioactive leucine or phenylalamine incorporated to cellular proteins were detected in the natural complex, with two associated enzymatic activities: one autolytic, the other endonucleolytic. The autolytic activity, known to be localized mainly in the cell wall and the endonucleolytic one, similar to the enzyme localized in cell membrane and periplasmic space of B. subtilis, suggest that donor DNA is complexed with a cell wall and/or a cell membrane fragment. Consideration of several characteristics of the natural complex: its density, protein content, and partial resistance of its DNA to DNase I, point to partial shielding of donor DNA in the cell fragment structure, and existence of a portion in a free uncovered form. Considerations on the possible role of the two enzymatic activities were based on the fact that they were not found in the complex formed by denatured DNA added to cells before lysis (reconstruction complex), and on hypotheses of their possible physioligical role.Part of the above results was presented in preliminary form at the Third European Meeting on Bacterial Transformation and Transfection, Granada, Spain, August 31–September 3, 1976     

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.