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.     

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.     

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.     

CodY is required for nutritional repression of Bacillus subtilis genetic competence.

The acquisition of genetic competence by Bacillus subtilis is repressed when the growth medium contains Casamino Acids. This repression was shown to be exerted at the level of expression from the promoters of the competence-regulatory genes srfA and comK and was relieved in strains carrying a null mutation in the codY gene. DNase I footprinting experiments showed that purified CodY binds directly to the srfA and comK promoter regions.     

Cell surface localization and processing of the ComG proteins, required for DNA binding during transformation of Bacillus subtilis

The comG operon of Bacillus subtilis encodes seven proteins essential for the binding of transforming DNA to the competent cell surface. We have explored the processing of the ComG proteins and the cellular localization of six of them. All of the proteins were found to be membrane associated. The four proteins with N-terminal sequence motifs typical of type 4 prepilins (ComGC, GD, GE and GG) are processed by a pathway that requires the product of comC , also an essential competence gene. The unprocessed forms of ComGC and GD behave like integral membrane proteins. Pre-ComGG differs from pre-ComGC and pre-ComGD, in that it is accessible to proteolysis only from the cytoplasmic face of the membrane and at least a portion of it behaves like a peripheral membrane protein. The mature forms of these proteins are translocated to the outer face of the membrane and are liberated when peptidoglycan is hydrolysed by lysozyme or mutanolysin. ComGG exists in part as a disulphide-cross-linked homodimer in vivo . ComGC was found to possess an intramolecular disulphide bond. The previously identified homodimer form of this protein is not stabilized by disulphide bond formation. ComGF behaves as an integral membrane protein, while ComGA, a putative ATPase, is located on the inner face of the membrane as a peripheral membrane protein. Possible roles of the ComG proteins in DNA binding to the competent cell surface are discussed in the light of these and other results.     

Genetic fate of DNA in a strain of Bacillus subtilis which is impaired in genetic transformation.

A mutation in Bacillus subtilis call recC4 which results in an impairment of genetic transformation was transferred to a new strain using the closely linked marker mit-2 (mitomycin C-resistance) for selection. This derived strain was in turn impaired in transformation but showed normal levels of sensitivity to ultraviolet irradiation and methyl methane sulfonate. The genetic and molecular fate of transforming DNA in the recC4 strain was studied. Normal amounts of DNA were taken up by the cells and this DNA or parts of it became associated with recipient DNA. Linkage between genes on donor and recipient molecules was, however, not established and transformants were not generated. The recC4 mutation therefore affects a step in the recombination pathway during transformation. Either the association between donor and recipient DNA molecules is abnormal or the cells are deficient in the further processing of the associated complex.     

TmrB protein, responsible for tunicamycin resistance of Bacillus subtilis, is a novel ATP-binding membrane protein.

tmrB is the gene responsible for tunicamycin resistance in Bacillus subtilis. It is predicted that an increase in tmrB gene expression makes B. subtilis tunicamycin resistant. To examine the tmrB gene product, we produced the tmrB gene product in Escherichia coli by using the tac promoter. TmrB protein was found not only in the cytoplasm fraction but also in the membrane fraction. Although TmrB protein is entirely hydrophilic and has no hydrophobic stretch of amino acids sufficient to span the membrane, its C-terminal 18 amino acids could form an amphiphilic alpha-helix. Breaking this potential alpha-helix by introducing proline residues or a stop codon into this region caused the release of this membrane-bound protein into the cytoplasmic fraction, indicating that the C-terminal 18 residues were essential for membrane binding. On the other hand, TmrB protein has an ATP-binding consensus sequence in the N-terminal region. We have tested whether this sequence actually has the ability to bind ATP by photoaffinity cross-linking with azido-[alpha-32P]ATP. Wild-type protein bound azido-ATP well, but mutants with substitutions in the consensus amino acids were unable to bind azido-ATP. These C-terminal or N-terminal mutant genes were unable to confer tunicamycin resistance on B. subtilis in a multicopy state. It is concluded that TmrB protein is a novel ATP-binding protein which is anchored to the membrane with its C-terminal amphiphilic alpha-helix.     

The membrane domain of SpoIIIE is required for membrane fusion during Bacillus subtilis sporulation

During Bacillus subtilis sporulation, SpoIIIE is required for both postseptational chromosome segregation and membrane fusion after engulfment. Here we demonstrate that SpoIIIE must be present in the mother cell to promote membrane fusion and that the N-terminal membrane-spanning segments constitute a minimal membrane fusion domain, as well as direct septal localization.     

Divalent metal is required for both phosphate transport and phosphate binding to phosphorin, a proteolipid isolated from brush-border membrane vesicles

The Na+-dependent phosphate transport system in the brush border of rabbit kidney exhibits a positive requirement for a divalent metal ion. Treatment of the brush-border membrane vesicles (BBMV) with a divalent metal chelator in combination with the divalent metal ionophore A23187 dramatically and selectively decreased the Na+-dependent uptake of phosphate; Na+-independent uptake of phosphate was not affected. The combination of chelator plus A23187 also inhibited uptake of phosphate in the presence of Na+ but in the absence of a gradient for sodium across the BBMV. This indicates that the inhibitor is not a result of an alteration in the Na+ gradient by chelator plus ionophore. The inhibited Na+ gradient-dependent transport of phosphate was restored by removing the chelator and adding Mn2+ to the BBMV. The phosphate-binding proteolipid (phosphorin) isolated from rabbit kidney BBMV binds inorganic phosphate with high affinity and specificity. Binding of phosphate to phosphorin is also inhibited by divalent metal chelators and can be restored by addition of a divalent metal. We conclude that a divalent metal ion is required both for the Na+-dependent phosphate transport in BBMV and for the binding of phosphate to the proteolipid phosphorin. These findings are consistent with our suggestion that phosphorin is a component of the Na+-dependent phosphate transport system in renal brush-border membranes.     

Mutation of the putative nucleotide binding site of the Bacillus subtilis membrane protein ComFA abolishes the uptake of DNA during transformation.

ComFA is a membrane protein required for the uptake of transforming DNA following its binding to the Bacillus subtilis competent-cell surface. ComFA, which resembles members of the DEAD family of ATP-driven helicases, contains sequences similar to those found in many ATP-binding proteins and thought to represent the ATP-binding sites of these proteins. We have suggested that ComFA may function as a DNA translocase and/or helicase, using the energy of ATP hydrolysis to mediate the uptake of DNA. As a partial test of this hypothesis, we have introduced mutations into highly conserved glycyl and lysyl residues of the putative ATP-binding site, located, respectively, at positions 151 and 152, and determined the effects of these alterations on in vivo function. A substitution of the conserved lysyl by a glutamyl residue (K152E) and a double G151R-K152N mutation each resulted in a nearly 1,000-fold decrease in transformability, equivalent to that observed in a ComFA null mutant. A K152N mutation caused a partial loss-of-function phenotype. These effects were manifested at the level of DNA uptake; no marked effects on the final levels of DNA binding were noted. When either the K152E mutant allele or the G151R-K152N double mutant allele was combined in single copy with wild-type comFA, a dominant negative phenotype expressed on the level of DNA uptake was observed, suggesting that ComFA acts in a complex with other proteins, with additional molecules of ComFA, or with both.     

Processing of a membrane protein required for cell-to-cell signaling during endospore formation in Bacillus subtilis

Activation of the late prespore-specific RNA polymerase sigma factor sigma(G) during Bacillus subtilis sporulation coincides with completion of the engulfment process, when the prespore becomes a protoplast fully surrounded by the mother cell cytoplasm and separated from it by a double membrane system. Activation of sigma(G) also requires expression of spoIIIJ, coding for a membrane protein translocase of the YidC/Oxa1p/Alb3 family, and of the mother cell-specific spoIIIA operon. Here we present genetic and biochemical evidence indicating that SpoIIIAE, the product of one of the spoIIIA cistrons, and SpoIIIJ interact in the membrane, thereby linking the function of the spoIIIJ and spoIIIA loci in the activation of sigma(G). We also show that SpoIIIAE has a functional Sec-type signal peptide, which is cleaved during sporulation. Furthermore, mutations that reduce or eliminate processing of the SpoIIIAE signal peptide arrest sporulation following engulfment completion and prevent activation of sigma(G). SpoIIIJ-type proteins can function in cooperation with or independently of the Sec system. In one model, SpoIIIJ interacts with SpoIIIAE in the context of the Sec translocon to promote its correct localization and/or topology in the membrane, so that it can signal the activation of sigma(G) following engulfment completion.     

An essential nuclear envelope integral membrane protein, Brr6p, required for nuclear transport

Despite rapid advances in our understanding of the function of the nuclear pore complex in nuclear transport, little is known about the role the nuclear envelope itself may play in this critical process. A small number of integral membrane proteins specific to the envelope have been identified in budding yeast, however, none has been reported to affect transport. We have identified an essential gene, BRR6, whose product, Brr6p, behaves like a nuclear envelope integral membrane protein. Notably, the brr6-1 mutant specifically affects transport of mRNA and a protein reporter containing a nuclear export signal. In addition, Brr6p depletion alters nucleoporin distribution and nuclear envelope morphology, suggesting that the protein is required for the spatial organization of nuclear pores. BRR6 interacts genetically with a subset of nucleoporins, and Brr6-green fluorescent protein (GFP) localizes in a punctate nuclear rim pattern, suggesting location at or near the nuclear pore. However, Brr6-GFP fails to redistribute in a (Delta)nup133 mutant, distinguishing Brr6p from known proteins of the pore membrane domain. We hypothesize that Brr6p is located adjacent to the nuclear pore and interacts functionally with the pore and transport machinery.     

An intron binding protein is required for transformation ability of p53.

Regulatory elements in intron sequences have been identified for several eukaryotic genes. The fourth intron of p53 is known to increase expression of p53 in a position dependent manner. We asked whether p53 intron 4 sequences interacted with DNA binding proteins to exact their effect. Three overlapping DNA fragments spanning the 5' end of p53 intron 4 were determined to specifically interact with protein in nuclear extracts from several cell lines by band shift analysis. Methylation interference experiments were used to identify purine residues involved in this protein-DNA interaction. Two G nucleotides were identified at intron 4 positions 33 and 44 and these were replaced by T and C, respectively. These two single base pair substitutions in the intron resulted in 1) lack of protein binding and 2) decreased expression of p53 as measured by a transformation assay. Thus the binding of protein to p53 intron 4 was shown to have functional significance. These experiments demonstrated a specific protein binding region in the 5' end of intron 4 critical for p53 expression and distinct from those elements already known to be involved in splicing.     

Transformation in Bacillus subtilis: a 75,000-dalton protein complex is involved in binding and entry of donor DNA.

A 75,000-dalton protein complex involved in DNA binding during transformation was purified from membranes of competent Bacillus subtilis cells. Previous results (Smith et al., J. Bacteriol. 156:101-108, 1983) showed that the complex contained two polypeptides, polypeptide a (molecular weight, 18,000; isoelectric point, 5.0) and polypeptide b (molecular weight, 17,000; isoelectric point, 4.7) in approximately equal amounts. In the present experiments the two polypeptides were extracted from two-dimensional gels and studied separately and in combination with respect to DNA binding and nuclease activities. For DNA binding the interaction of both polypeptides was required. DNA binding occurred efficiently in the presence of EDTA. Nuclease activity was restricted to polypeptide b. The nucleolytic properties of b were identical to those of the native 75,000-dalton complex. Polypeptide a affected b by reducing its nuclease activity. Analysis of the nuclease subunit b on DNA-containing polyacrylamide gels revealed nuclease activities at four different molecular weight positions. These activities were identical to the major competence-specific nuclease activities which were previously implicated in the entry of donor DNA during transformation (Mulder and Venema, J. Bacteriol. 152:166-174, 1982). These results indicate that the 75,000-dalton protein complex is composed of two different competence-specific polypeptides involved in both binding and entry of donor DNA. The possible roles of the two polypeptides in the transformation of B. subtilis are discussed.     

Plasmid DNA in a groundwater aquifer microcosm -adsorption, DNAase resistance and natural genetic transformation of Bacillus subtilis

Prokaryotes can exchange chromosomal and plasmid genes via extracellular DNA in a process termed genetic transformation. This process has been observed in the test tube for several bacterial species living in the environment but it is not clear whether transformation occurs in natural bacterial habitats. A major constituent of terrestrial environments are solid particles such as quartz, silt and clay, which have considerable surface areas and which make up the solid-liquid interfaces of the habitat. In previous experiments the adsorption of DNA to chemically purified quartz and clay minerals was shown and the partial protection of adsorbed DNA against DNAase I. In a microcosm consisting of natural groundwater aquifer material (GWA) sampled directly from the environment and groundwater (GW) both linear duplex and supercoiled plasmid DNA molecules bound rapidly and quantitatively to the minerals. The divalent cations required to form the association were those present in the GWA/GW microcosm. The association was stable to extended elution over one week at 23°C. Upon adsorption, the DNA became highly resistant against enzymatic degradation. About 1000 times higher DNAase I concentrations were needed to degrade bound DNA to the same extent as DNA dissolved in GW. Furthermore, chromosomal and plasmid DNA bound on GWA transformed competent cells of Bacillus subtilis. However, in contrast to DNA in solution, on GWA the chromosomal DNA was more active in transformation than the plasmid DNA. The studies also revealed that in the transformation of B. subtilis Mg²⁺ can be replaced by Na⁺, K⁺ or NH₄ The observations suggest that in soil and sediment environments, mineral material with inorganic precipitates and organic matter can harbour extracellular DNA leaving it available for genetic transformation.     

Bacillus subtilis CheD is a chemoreceptor modification enzyme required for chemotaxis

The chemotaxis machinery of Bacillus subtilis is similar to that of the well characterized system of Escherichia coli. However, B. subtilis contains several chemotaxis genes not found in the E. coli genome, such as cheC and cheD, indicating that the B. subtilis chemotactic system is more complex. In B. subtilis, CheD is required for chemotaxis; the cheD mutant displays a tumbly phenotype, has abnormally methylated chemoreceptors, and responds poorly to most chemical stimuli. Homologs of B. subtilis CheD have been found in chemotaxis-like operons of a large number of bacteria and archaea, suggesting that CheD plays an important role in chemotactic sensory transduction for many organisms. However, the molecular function of CheD has remained unknown. In this study, we show that CheD catalyzes amide hydrolysis of specific glutaminyl side chains of the B. subtilis chemoreceptor McpA. In addition, we present evidence that CheD deamidates other B. subtilis chemoreceptors including McpB and McpC. Previously, deamidation of B. subtilis receptors was thought to be catalyzed by the CheB methylesterase, as is the case for E. coli receptors. Because cheD mutant cells do not respond to most chemoattractants, we conclude that deamidation by CheD is required for B. subtilis chemoreceptors to effectively transduce signals to the CheA kinase.