Development Of Genetic Competence Research Articles

ComA-dependent transcriptional activation of lichenysin A synthetase promoter in Bacillus subtilis cells.

ComA is a DNA-binding activator protein which is required for the transcription of several late-growth phase expressed genes including srfA, an operon needed for the development of genetic competence, efficient sporulation, and surfactin production in Bacillus subtilis (B. subtilis). We show here that the ComA protein can also recognize the promoter regulatory region of the lchA, lichenysin A synthetase operon, found in. Bacillus licheniformis (B. licheniformis) when introduced into B. subtilis cells. Mutational analysis of this region suggests that a palindromic sequence upstream of the lchA promoter may be the target for ComA-dependent transcriptional activation. Considering that the comA operon is present in the B. licheniformis chromosome, we propose the similar mechanism of transcriptional activation of the lichenysin A synthetase operon.

Analysis of non-polar deletion mutations in the genes of the spo0K ( opp) operon of Bacillus subtilis

The spo0K ( opp) operon of Bacillus subtilis encodes an oligopeptide permease that is required for uptake of oligopeptides, development of genetic competence, and initiation of sporulation. We made in-frame, non-polar deletion mutations in each of the first four genes of the five-gene spo0K operon and tested effects on oligopeptide transport, sporulation, and expression of competence genes. spo0KA, B, C, and D were required for sporulation, competence development, and oligopeptide transport. Disruption of spo0KE caused a less severe phenotype than did disruption of any of the other genes of the operon.

Quorum sensing by peptide pheromones and two-component signal-transduction systems in Gram-positive bacteria.

Cell-density-dependent gene expression appears to be widely spread in bacteria. This quorum-sensing phenomenon has been well established in Gram-negative bacteria, where N-acyl homoserine lactones are the diffusible communication molecules that modulate cell-density-dependent phenotypes. Similarly, a variety of processes are known to be regulated in a cell-density- or growth-phase-dependent manner in Gram-positive bacteria. Examples of such quorum-sensing modes in Gram-positive bacteria are the development of genetic competence in Bacillus subtilis and Streptococcus pneumoniae, the virulence response in Staphylococcus aureus, and the production of antimicrobial peptides by several species of Gram-positive bacteria including lactic acid bacteria. Cell-density-dependent regulatory modes in these systems appear to follow a common theme, in which the signal molecule is a post-translationally processed peptide that is secreted by a dedicated ATP-binding-cassette exporter. This secreted peptide pheromone functions as the input signal for a specific sensor component of a two-component signal-transduction system. Moreover, genetic linkage of the common elements involved results in autoregulation of peptide-pheromone production.

A regulatory switch involving a Clp ATPase.

Clp ATPase chaperone proteins are found in procaryotes and eucaryotes. Recently, ClpC of Bacillus subtilis was found to be part of a regulatory switch(1). ClpC, in combination with the MecA and ComS proteins, regulates the activity of a transcription factor, ComK, which is necessary for the development of genetic competence (the ability to bind and take up exogenous DNA). The complex of ClpC:MecA:ComK renders ComK inactive. Interaction between ComS and the ternary complex releases active ComK. This regulatory switch controls ComK activity in response to cell density signals that affect production of ComS. Regulated interaction between Clp ATPase and target proteins might prove to be widespread.

Biochemical characterization of a molecular switch involving the heat shock protein ClpC, which controls the activity of ComK, the competence transcription factor of Bacillus subtilis.

Development of genetic competence in Bacillus subtilis is controlled by the competence-specific transcription factor ComK. ComK activates transcription of itself and several other genes required for competence. The activity of ComK is controlled by other genes including mecA, clpC, and comS. We have used purified ComK, MecA, ClpC, and synthetic ComS to study their interactions and have demonstrated the following mechanism for ComK regulation. ClpC, in the presence of ATP, forms a ternary complex with MecA and ComK, which prevents ComK from binding to its specific DNA target. This complex dissociates when ComS is added, liberating active ComK. ClpC and MecA function as a molecular switch, in which MecA confers molecular recognition, connecting ClpC to ComK and to ComS.

Plasmid-amplified comS enhances genetic competence and suppresses sinR in Bacillus subtilis.

The establishment of genetic competence in Bacillus subtilis is controlled by a vast signal transduction network involving the products of genes that function in several postexponential-phase processes. Two of these proteins, SinR and DegU, serve as molecular switches that influence a cell's decision to undergo either sporulation or genetic competence development. In order to determine the roles of SinR and DegU in competence control, multicopy suppression experiments with plasmid-amplified comS, SinR, and degU genes were undertaken. Multicopy comS was found to elevate competence gene transcription and transformation efficiency in both wild-type and sinR mutant cells but not in degU mutant cells. Multicopy degU failed to suppress comS or sinR mutations. No suppression of comS or degU by multicopy sinR was observed. The expression of a comS'::'lacZ translational fusion and srf-lacZ operon fusion was examined in sinR cells and cells bearing plasmid-amplified sinR. The expression of comS'::'lacZ gene fusion was reduced by the sinR mutation, but both comS'::'lacZ and srf-lacZ were repressed by multicopy sinR. Cells bearing plasmid-amplified sinR were poorly competent. These results suggest that sinR is required for optimal comS expression but not transcription from the srf promoter and that SinR at high concentrations represses srf transcription initiation.

Characterization of the Haemophilus influenzae topA locus: DNA topoisomerase I is required for genetic competence

A gene essential for the development of genetic competence in Haemophilus influenzae (Hi) was identified as a homolog of the Escherichia coli (Ec) topA gene, which encodes DNA topoisomerase I (TopI). The Hi topA locus was initially identified by mini-Tn lOkan mutagenesis. Three independent insertion events within 500 bp of each other resulted in mutant strains that shared a similar phenotype. Each was deficient in competence-induced DNA binding, showed increased sensitivity to UV irradiation, and had an increased doubling time as compared to the wild-type (wt) strain. The nucleotide sequence of a 6.6-kb fragment containing the wt allele was determined. The sequence contained an open reading frame (ORF) of 868 amino acids (aa) that was interrupted by each of the mini-Tn 10kan mutations. The deduced aa sequence had a molecular mass of 98 155 Da, a p I of 8.59 and showed strong similarity to Ec TopI. Examination of the topoisomer distribution of a test plasmid in an Hi mutant carrying an insertion in this ORF showed an increase in the level of supercoiling, indicating that TopI is necessary to relax supercoiled DNA in Hi. Complementation studies and insertional inactivation of genes downstream from topA indicated that TopI and not some downstream gene product was essential for competence. Four other ORFs were identified and two of these had homology to known genes. ORF1, which was truncated at one end of the sequenced region, shared strong sequence similarity to the C-terminal end of Ec pyridine nucleotide transhydrogenase β subunit. ORF4, which was also truncated, showed strong sequence similarity to the N-terminal end of Ec threonyl-tRNA synthetase.

Genetic networks controlling the initiation of sporulation and the development of genetic competence in Bacillus subtilis.

Interconnected regulatory networks control the initiation of sporulation and the development of genetic competence in Bacillus subtilis. These two developmental pathways have both common and distinct elements and employ similar regulatory strategies. Activation of the ComK transcription factor serves to integrate many of the physiological signals that control competence development, including cell density signals. The cell density signals for competence are mediated by two different peptide pheromones, the ComX pheromone, a 9 or 10 amino acid peptide with a modified tryptophan residue, and the competence stimulating factor, CSF, which is at least in part a peptide. Activation of the Spo0A transcription factor by phosphorylation serves as a developmental checkpoint and to integrate several physiological signals that control entry into the sporulation pathway. The physiological signals are generated by conditions of nutrient deprivation, high cell density, the Krebs cycle, DNA replication, DNA damage, and some aspect of the chromosome partitioning machinery. Both the ComK and Spo0A transcription factors are part of autogenous regulatory loops that control entry into competence or sporulation.

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.

Translation of the open reading frame encoded by comS, a gene of the srf operon, is necessary for the development of genetic competence, but not surfactin biosynthesis, in Bacillus subtilis.

A small open reading frame, comS of the srf operon, is the site of mutations that impair competence development in Bacillus subtilis. comS open reading frame translation was required for competence, as was confirmed by the suppression of a comS amber mutation [comS(Am)] by the nonsense suppressor sup-3. comS(Am), when introduced into the srf operon, eliminated late competence gene expression but had no significant effect on surfactin production.

The major role of Spo0A in genetic competence is to downregulate abrB, an essential competence gene

We show that the major role for Spo0A in the development of genetic competence is to downregulate expression of abrB. AbrB is both a negative regulator and a positive regulator of competence. The negative effects are exerted at multiple points in competence regulation. A regulatory mechanism that is independent of mecA and abrB operates on comK expression.

Identification of a DNA transformation gene required for com101A+ expression and supertransformer phenotype in Haemophilus influenzae.

DNA sequencing, RNA mapping, and protein expression experiments revealed the presence of a gene, tfoX+, encoding a 24.9-kDa polypeptide, that is transcribed divergently from a common promoter region with the Haemophilus influenzae rec-1+ gene. H. influenzae strains mutant for tfoX failed to bind transforming DNA and were transformation deficient. Primer extension experiments utilizing in vivo total RNA from precompetent and competent H. influenzae cells demonstrated that transcription of tfoX+ increased immediately upon competence induction, suggesting that tfoX+ is an early competence gene. Similar experiments showed that the expression of the late competence-specific gene, com101A+, was tfoX+ dependent. Moreover, expression of plasmid-borne tfoX+ in H. influenzae resulted in constitutive competence. The addition of cyclic adenosine monophosphate (cAMP) to strains carrying a tfoX::lacZ operon fusion resulted in an immediate increase in beta-galactosidase activity that correlated with an increase in genetic transformability. Collectively, our results suggest that TfoX may play a key role in the development of genetic competence by regulating the expression of late competence-specific genes.

Convergent sensing pathways mediate response to two extracellular competence factors in Bacillus subtilis.

Development of genetic competence in Bacillus subtilis is regulated by extracellular signaling molecules, including the ComX pheromone, a modified 9- or 10-amino-acid peptide. Here, we present characterization of a second extracellular competence stimulating factor (CSF). CSF appears to be, at least in part, a small peptide of between 520 and 720 daltons. Production of CSF requires several genes that are needed both for initiation of sporulation and development of competence (spo0H, spo0A, spo0B, and spo0F). Although both peptide factors regulate competence, two different sensing pathways mediate the response to the ComX pheromone and CSF. Analysis of double mutants indicated that ComX pheromone is on the same genetic pathway as the membrane-bound histidine protein kinase encoded by comP and that CSF is on the same genetic pathway as the oligopeptide permease encoded by spo0K. Furthermore, the cellular response to partly purified ComX pheromone requires the ComP histidine protein kinase, whereas the response to partly purified CSF requires the Spo0K oligopeptide permease. These two sensing pathways converge to activate competence genes. Both factors and their convergent sensing pathways are required for normal development of competence and might function to integrate different physiological signals.

Genetic Networks Controlling the Initiation of Sporulation and the Development of Genetic Competence in Bacillus subtilis

Genetic Networks Controlling the Initiation of Sporulation and the Development of Genetic Competence in Bacillus subtilis

ComK acts as an autoregulatory control switch in the signal transduction route to competence in Bacillus subtilis

The comK gene is a regulatory transcription unit which is essential for the development of genetic competence in Bacillus subtilis. The transcription of comK is under strict nutritional and growth phase-dependent control and has been shown to depend on the gene products of comA and srfA. In this report, we show that expression of comK is dependent on its own gene product as well as on the gene products of all other tested regulatory genes known to be involved in competence development (abrB, comA, comP, degU, sin, spo0A, spo0H, spo0K, and srfA). A mecA mutation is able to suppress the competence deficiency of mutations in any of these regulatory loci except for mutations in spo0A and, as we show here, in comK. Furthermore, we show that the presence of comK on a multiple copy plasmid leads to derepression of comK expression, causing an almost constitutive expression of competence in minimal medium as well as permitting competence development in complex medium. We infer from these results that the signals which trigger competence development, after having been received and processed by the various components of the competence signal transduction pathway, all converge at the level of comK expression. As soon as derepression of comK expression occurs, the positive autoregulation rapidly results in accumulation of the comK gene product, which subsequently induces competence.

Activation of spo0A transcription by sigma H is necessary for sporulation but not for competence in Bacillus subtilis.

spo0A and spo0H are needed for the initiation of sporulation and for the development of genetic competence in Bacillus subtilis. Transcription of spo0A initiates from two promoters, Pv and Ps. Pv is active during vegetative growth and is recognized by RNA polymerase containing sigma A. Expression from Ps increases during sporulation and depends on sigma H, the spo0H gene product. A deletion mutation, spo0A delta Ps, that removes the promoter controlled by sigma H blocked sporulation but had no detectable effect on competence. These results indicate that expression of spo0A from Ps is necessary for sporulation and that the requirement for spo0H in competence development is not due to its role in expression of spo0A.

Biochemical and genetic characterization of a competence pheromone from B. subtilis

We have purified and characterized a modified peptide pheromone that accumulates in culture medium as B. subtilis grows to high density. This pheromone is required for the development of genetic competence. When added to cells at low density, the pheromone induces the premature development of competence. The peptide moiety of the pheromone matches nine of the last ten amino acids predicted from a 55 codon open reading frame, comX. comX and comQ, the gene immediately upstream of comX, are required for production of the pheromone. Response to the pheromone requires the comP-comA two-component regulatory system and the oligopeptide permease encoded by spo0K. Spo0K could transport the pheromone into the cell, or function as a receptor, binding the pheromone and sending a transmembrane signal, leading to activation of the ComA transcription factor and induction of competence development.

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.

Regulation of peptide antibiotic production in Bacillus

In Bacillus species, starvation leads to the activation of a number of processes that affect the ability to survive during periods of nutritional stress. Activities that are induced include the development of genetic competence, sporulation, the synthesis of degradative enzymes, motility, and antibiotic production. The genes that function in these processes are activated during the transition from exponential to stationary phase and are controlled by mechanisms that operate primarily at the level of transcription initiation. One class of genes functions in the synthesis of special metabolites such as the peptide antibiotics tyrocidine and gramicidin S as well as the cyclic lipopeptide surfactin. These genes include the grs and tyc operons in Bacillus brevis, which encode gramicidin S synthetase and tyrocidine synthetase, respectively, and the srfA operon of Bacillus subtilis which encodes the enzymes of the surfactin synthetase complex. Peptide antibiotic biosynthesis genes are regulated by factors as diverse as the early sporulation gene product Spo0A, the transition-state regulator AbrB, and gene products (ComA, ComP, and ComQ) required for the initiation of the competence developmental pathway.

Modification in penicillin-binding proteins during in vivo development of genetic competence of Haemophilus influenzae is associated with a rapid change in the physiological state of cells.

By using whole-cell labeling assay with 125I-penicillin V, we observed a reduction in the binding of the radiolabeled beta-lactam to four or five penicillin-binding proteins (PBPs) in Haemophilus influenzae cells cultivated under specific conditions. PBPs 3A, 3B, 4, and 6 were altered after the growth of bacteria in diffusion chambers implanted in the peritoneal cavity of rats. PBP 2 was also modified when cells were cultivated in human cerebrospinal fluids. Because this observation may have important consequences on the efficacy of beta-lactams during antibiotic therapy, we characterized the physiological state of bacteria cultivated in animals in the hope of explaining how such important changes in cell properties develop in vivo. Since the development of natural genetic competence occurs at the stationary phase of growth in H. influenzae, we used a DNA transformation assay to evaluate the physiological state of bacteria grown in diffusion chambers implanted in rats. Chromosomal DNA isolated from an antibiotic-resistant donor strain was mixed with bacteria in diffusion chambers. At different times during a 5-h incubation period, recipient bacteria were collected from the chambers, CFU were determined by plate counting, and antibiotic-resistant transformants were isolated on selective plates. Genetic competence rapidly developed in cells grown in rats, and the frequency of transformation by test DNA was elevated. Electron microscopy revealed an irregular cell shape and blebs at the surface of bacteria cultivated in animals and in cerebrospinal fluids. In an attempt to induce a similar physiological state in vitro, we supplemented broth cultures with cyclic AMP or synchronized cultures by a nutritional upshift. No changes in PBPs were observed with supplemental cyclic AMP or during a single cell cycle. Finally, a reduction in the affinity of PBPs for 125I-penicillin V identical to that observed in bacteria grown in rats was observed in cells isolated from the stationary phase of growth in vitro. These results clearly indicate that H. influenzae cells grown in animals undergo a rapid change to a physiological state similar to that found in late-stationary-phase cultures in vitro. This observation indicates that the rational design of future and improved antibiotic therapy of H. influenzae infections should consider cell properties of slow-growing or latent bacteria.