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D Dubnau

Publications and source records attributed to D Dubnau.

At least 37 records · Page 2Linked to original sources
PubMed1996

Regulatory inputs for the synthesis of ComK, the competence transcription factor of Bacillus subtilis.

Competence in Bacillus subtilis is expressed post-exponentially in response to signals which are interpreted by a complex network of regulatory proteins. This network culminates in the transcriptional activation of a set of late-competence proteins that mediate DNA binding and uptake during transformation. ComK, a protein that binds to competence promoters and appears to activate their transcription, is itself synthesized in response to the signal-transduction network. ComK is known to be required for the transcription of its own gene. We have placed comK under control of the xylose-inducible PxylA promoter and used this construct to show that ComK synthesis is sufficient as well as necessary to induce competence. We have also confirmed that the Mec proteins act post-transcriptionally to inactivate ComK, probably by protein-protein interaction. We have further demonstrated that ComS is required to generate an upstream signal that causes reversal of Mec-induced inactivation of ComK. In addition to ComK itself, DegU, AbrB, and SinR are required for comK transcription; mutations in their genes are bypassed by PxylA-comK induction, and therefore their products appear not to act via the Mec proteins. Overproduction of ComK, in a loss-of-function mec mutant, is also known to bypass the need for DegU, SinR and AbrB. We propose that these proteins enhance the activity of ComK as a positive autoregulatory transcription factor, acting as coactivator proteins when ComK is present at low concentrations. Finally, we demonstrate that when ComK is synthesized from the PxylA promoter and mecA is inactivated by mutation, no additional growth-stage-regulated control of competence can be detected.

Bacillus subtilis↗
PubMed1996

Polynucleotide phosphorylase is necessary for competence development in Bacillus subtilis.

comR (pnpA) is a newly identified gene in Bacillus subtilis that is necessary for the expression of late competence genes. Transformability of a comR (pnpA) mutant is 1-5% of that seen in comR+ strains. Cloning and sequencing identified ComR as polynucleotide phosphorylase (PNPase). The PNPase amino acid sequence has 50% identity and 67% similarity with the Escherichia coli enzyme. Enzymatic assays show that this is the only PNPase activity in B. subtilis. comR (pnpA) is necessary for comG-lacZ and comK-lacZ expression, but this requirement is bypassed by a mecA disruption. In B. subtilis, the loss of PNPase has little effect on expression from a fusion of the srfA promoter directly to lacZ, but is necessary for normal expression from certain srfA-lacZ fusions that include portions of the normal srfA transcript. When a srfA-lacZ translational fusion is tested in isogenic pnpA+ and pnpA derivatives of E. coli, lower expression is seen in the pnpA mutant. Since expression from lacZ fusions to comA, sinR, and mecA appeared similar in the B. subtilis pnpA and pnpA+ strains, the loss of PNPase does not have a strong general effect on gene expression. These results suggest that PNPase may be necessary for modification of the srfA transcript in order to activate translation or stabilize the transcript, and that this may be necessary for competence development. This is the first evidence of post-transcriptional effects on the development of competence in B. subtilis.

Amino Acid Sequence↗
PubMed1995

Inactivation of mecA prevents recovery from the competent state and interferes with cell division and the partitioning of nucleoids in Bacillus subtilis.

The development of genetic competence in Bacillus subtilis requires the synthesis of ComK, a transcription factor, which is normally produced as a culture enters the stationary phase. This synthesis is known to be regulated in part by the protein MecA. Loss-of-function mutations in mecA result in overexpression of ComK and its appearance early during exponential growth. We show here that mecA inactivation also causes a loss of colony-forming ability, especially during stationary phase. This loss is accompanied by the appearance of cells in which normal nucleoid separation has failed to occur. Renografin gradient fractionation of mecA cultures grown to competence reveals that nearly 100% of the cells band at the low buoyant density characteristic of competent cells, and that this low density is competence-related. The loss of viability, the low buoyant density and the nucleoid separation defect, are all comK-dependent. The loss of viability can be reversed by even the transient introduction of mecA+. It is proposed that these effects of ComK overexpression are related to the DNA replication arrest normally exhibited by the competent cell fraction and that MecA is needed to reverse this arrest and to permit escape from the competent state. The shift of nearly 100% of the cells to light buoyant density in a mecA mutant culture strongly suggests that the MecA protein is a regulator of the cell-type-specific expression of competence.

Bacillus subtilis↗
PubMed1995

comK encodes the competence transcription factor, the key regulatory protein for competence development in Bacillus subtilis.

comK is a positive autoregulatory gene occupying a central position in the competence-signal-transduction network. All regulatory routes identified in this network converge at the level of comK expression. The ComK protein is required for the transcriptional induction of comK and the late competence genes, which specify morphogenetic and structural proteins necessary for construction of the DNA-binding and uptake apparatus. In this report we demonstrate that ComK specifically binds to DNA fragments containing promoter and upstream sequences of the genes it affects (comC, comE, comF, comG and comK). Using portions of the region upstream of comC we show that the ComK-binding sequences are essential for the expression of competence. Moreover, we demonstrate that the presence of ComK stimulates the expression of comF-lacZ and comG-lacZ translational fusions in vivo in Escherichia coli. These results indicate that the gene product of comK is identical to the previously inferred competence transcription factor (CTF).

Amino Acid Sequence↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1994

Regulation of competence-specific gene expression by Mec-mediated protein-protein interaction in Bacillus subtilis.

The expression of competence genes in Bacillus subtilis is controlled by a signal transduction cascade which increases the expression of a competence transcription factor (CTF, encoded by comK) during the transition from exponential growth to stationary phase. The transcription of CTF (ComK) is decreased by the product of the mecA gene, and this inhibition is relieved in response to an unknown signal received from upstream in the regulatory pathway. Inactivation of either mecA or another gene, mecB, results in overproduction of ComK. We show here that the concentration of MecA protein does not vary markedly with culture medium, as a function of growth stage, or in competent and noncompetent cells. We also show that MecA can interact directly with ComK. Finally, evidence is presented suggesting that MecB functions prior to MecA in the signaling pathway. A model is discussed which involves the sequestration of ComK by MecA binding and the release of the transcription factor when an appropriate signal is relayed to MecA by MecB.

Amino Acid Sequence↗
PubMed1994

Membrane association and role in DNA uptake of the Bacillus subtilis PriA analogue ComF1.

The late competence protein ComF1 is required for genetic transformation in Bacillus subtilis. Because of the sequence similarities of ComF1 to known ATP-dependent DNA helicases and translocases, we have hypothesized that this protein either unwinds bound double-stranded DNA or helps in the translocation of the transforming single-stranded DNA across the cell membrane. Two important implications of this hypothesis (the association of ComF1 with the membrane and its specific requirement for DNA uptake) have been tested in this report. Using cell fractionation techniques and Western blotting analysis, we show that ComF1 is located almost exclusively on the cell membrane and that it is membrane-targeted independently of other competence proteins. Moreover, ComF1 behaves like an integral membrane protein in extractability and detergent partition assays. We also show that this protein is required for the DNA-uptake step during transformation but not for DNA binding to the cell surface. DNA uptake is blocked in strains with null mutations or in-frame deletions in comF1 but also in strains that overproduce the ComF1 protein under competence conditions. This last observation suggests that ComF1 expression must be balanced with that of other competence proteins, with which it may interact to form a multisubunit complex for DNA uptake.

Amino Acid Sequence↗
PubMed1994

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.

Bacillus subtilis↗
PubMed1994

The regulation of competence transcription factor synthesis constitutes a critical control point in the regulation of competence in Bacillus subtilis.

comK, which encodes the competence transcription factor, is itself transcriptionally activated at the transition from exponential growth to stationary phase in Bacillus subtilis. MecA, a negative regulator of competence, also inhibits comK transcription when overexpressed, and a mecA null mutation results in comK overexpression. Although null mutations in mecA, as well as in another gene, mecB, are known to bypass the requirements for nearly all of the competence regulatory genes, the comK requirement is not suppressed by mecA inactivation. Various competence regulatory genes (comA, srfA, degU, abrB, sin, and spo0A) are shown to be required for the expression of comK. srfA transcription is shown to occur equally in cells destined for competence and those destined not to become competent. In contrast, comK transcription is restricted to the presumptive competent cells. These and other results are combined to describe a regulatory pathway for competence.

Bacillus subtilis↗
PubMed1993

Characterization of comE, a late competence operon of Bacillus subtilis required for the binding and uptake of transforming DNA.

The binding and transport of DNA by competent Bacillus subtilis requires the assembly of a specialized apparatus. We present here the characterization of comE, an operon under competence control that is required for both DNA binding to the competent cell surface, and for uptake. comE contains three open reading frames (ORF1-3) read in the forward direction, preceded by a long untranslated leader sequence and an apparent E sigma A promoter. A minor promoter also is responsible for transcription of ORF2 and -3. A transcript containing a single ORF is produced in the reverse direction. The reverse ORF overlaps ORF1 and the untranslated comE leader. The comE transcript is present at a very low level during growth and at an elevated level in stationary-phase cells. Conversely, the reverse transcript is present during exponential growth and disappears during stationary phase. The reverse ORF resembles prokaryotic and eukaryotic pyrroline-5'-carboxylate reductases, while ORF2 is similar to several dCMP deaminases. ORF1 and ORF3 are predicted to be integral membrane proteins. The latter is specifically required for DNA uptake but not for binding.

Amino Acid Sequence↗
PubMed1993

comF, a Bacillus subtilis late competence locus, encodes a protein similar to ATP-dependent RNA/DNA helicases.

We have sequenced and genetically characterized comF, a Bacillus subtilis competence locus, previously identified by Tn917 transposon insertion mutagenesis. Expression of the locus, in which three open reading frames (ORFs) were found, is driven by a single sigma A-like promoter in front of comFORF1 and is dependent on early regulatory competence genes and only expressed in competence medium. The predicted amino acid sequences of two of the ORFs showed similarities to known proteins in the GenBank and SwissProt databases: ComFORF1 is similar to an extensive family of ATP-dependent RNA/DNA helicases with closer similarity to the DEAD protein subfamily and to the PriA protein in Escherichia coli. The latter is a DNA translocase/helicase required for primosome assembly at the replication fork of phage phi X174. ComFORF3 is 22% identical to Com101, a protein required for genetic competence in Haemophilus influenzae, a naturally competent Gram-negative bacterium. In-frame comFORF1 deletions were 1000-fold deficient in transformability compared to the wild-type, whereas disruptions of the other two ORFs were only five- to 10-fold lower. These observations allow us to hypothesize that the ComFORF1 late gene product plays an essential role during the binding and uptake events involved in Bacillus subtilis transformation.

Adenosine Triphosphatases↗
PubMed1993

Sequence and properties of mecA, a negative regulator of genetic competence in Bacillus subtilis.

The development of competence in Bacillus subtilis is regulated by growth conditions and several regulatory genes. In complex media competence development is poor, and there is little or no expression of late competence genes. mec mutations permit competence development and late competence gene expression in complex media, and bypass the requirements for many of the competence regulatory genes. In this paper we describe the cloning and characterization of mecA. The mecA gene product acts negatively in the development of competence. Null mutations in mecA allowed expression of a late competence gene comG, under conditions where it is not normally expressed, including in complex media and in cells mutant for several competence regulatory genes. Overexpression of MecA from a multicopy plasmid resulted in inhibition of comG transcription. The DNA sequence of mecA was determined and the predicted gene product showed no significant similarity to any protein in the database. Expression of a mecA-lacZ translational fusion was constitutive during growth and did not vary significantly in the different media tested. The role of mecA in competence development and other stationary phase phenomena is discussed.

Amino Acid Sequence↗
PubMed1993

ComA, a phosphorylated response regulator protein of Bacillus subtilis, binds to the promoter region of srfA.

ComA is a response regulator protein of Bacillus subtilis which is required for the transcription of several genes which are involved in late-growth expression and in responses to environmental stress. Among these genes are degQ, gsiA, and srfA. The last is an operon needed for the development of genetic competence, surfactin production, and normal sporulation. We show here that partially purified ComA protein, isolated from an overproducing Escherichia coli strain, is phosphorylated in vitro by incubation with acetyl phosphate and that ComA could bind specifically to a DNA fragment containing the promoter of srfA and associated sequences. The binding affinity is enhanced when ComA is phosphorylated. DNase I protection analysis identified two protected sites located upstream from the srfA promoter. The presence of DNase I-hypersensitive bonds induced by ComA binding which are located between the protected sequences is consistent with a model for ComA action involving the bending of DNA.

Bacillus subtilis↗
PubMed1991

The regulation of genetic competence in Bacillus subtilis.

Genetic competence develops as a global response of Bacillus subtilis to the onset of stationary phase, in glucose-minimal salts-based media. The onset of competence is accompanied by the expression of several late gene products that are required for the binding, processing and uptake of transforming DNA. A number of regulatory genes have been identified that are needed for the appropriate synthesis of the late gene products. The regulatory gene products include a number of known transcription factors, as well as several members of the bacterial two-component regulatory system. Genetic analysis has suggested a scheme for the flow of regulatory information signalling the onset of competence. Most of these regulatory products appear to be involved in the response to nutritional status, while the components responsible for growth stage and cell-type-specific control remain unknown. The general implications of this scheme for post-exponential expression are discussed.

Bacillus subtilis↗
PubMed1991

Sequence and properties of comQ, a new competence regulatory gene of Bacillus subtilis.

The sequence and properties of the comQ gene are described. comQ was predicted to encode a 34,209-Da protein, and the product of comQ was shown to be required for the development of genetic competence. The apparent transcriptional initiation and termination sites of comQ were mapped, and the location of a likely E sigma A promoter was inferred. The expression of comQ was maximal early in growth and declined as the cells approached the stationary phase. This expression was not dependent on any of the competence regulatory genes tested (comA, comP, sin, abrB, degU, and spo0A). Disruption of comQ in the chromosome prevented the development of competence as well as the transcription of comG, a late competence operon. This disruption also decreased the expression of srfA, a regulatory operon needed for the expression of competence. These and other results suggest a role for ComQ early in the hierarchy of competence regulatory genes, probably as a component of a signal transduction system.

Amino Acid Sequence↗
PubMed1991

Growth stage signal transduction and the requirements for srfA induction in development of competence.

srfA is an operon needed for the development of genetic competence in Bacillus subtilis. This operon is normally expressed at a low level during growth, and its transcription increases sharply just before the transition to stationary phase. The genetic requirements for the full expression of srfA were previously examined in several laboratories and shown to include spo0A, spo0H, spo0K, comQ, and comA. In the present study these results were confirmed with an isogenic set of strains. We have also shown that comP is needed for srfA expression but that other regulatory genes required for competence (degU, sin, and abrB) are not needed for the expression of srfA. We have used the expression of srfA under control of the regulatable Pspac promoter to study the kinetics of competence development and to determine whether the genes ordinarily required for expression of srfA are needed for any additional roles during the development of competence. When expression of srfA was driven from Pspac, competence was expressed constitutively throughout growth. Furthermore, when srfA was expressed from Pspac, the spo0K, comQ, comP, and comA determinants were no longer required for the expression of competence. We conclude therefore that the multiple signals which trigger the initiation of competence development in relation to growth stage are ordinarily received prior to the increase in srfA expression. We propose that these signals are mediated by the products of spo0K, comQ, comP, and comA, resulting in the phosphorylation of ComA by ComP. This in turn would enable ComA to function as a positive transcription factor for srfA, leading to the elaboration of the srfA product(s) and the consequent initiation of competence. We also propose that this is the major, and possibly the only, role for the spo0K, comQ, comP, and comA products during competence development.

Bacillus subtilis↗