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PubMed1961

Studies on transformations of Hemophilus influenzae. I. Competence.

A procedure has been developed for obtaining Hemophilus influenzae of such competence that 1 to 10 per cent transform to any of several genetic factors by utilizing a period of aerobic growth followed by a non-aerobic period. Differences in levels of competence were not due to differences in genetic background. Competence was due to at least one factor intrinsic to the cell or site on the cell and was not transferable to non-competent cells. Competence was affected by salt concentration, pH, and temperature. Washing competent cells reduces their ability to transform, but not their capacity to bind DNA reversibly. The irreversible step could be restored with little or no accompanying growth. These facts suggest that reversible and irreversible binding represent separate biochemical steps. DNA initiates a reaction in cells leading to a loss of competence. In the absence of DNA the cells remain competent for at least an hour. Competence correlates quantitatively with predictability of multiple transformations. The observed and calculated values of multiple transformations are in closer agreement, the higher the frequency of transformation for single markers. The correction needed to bring the two figures into agreement is a measure of the fraction of non-competent cells.

DNA, Bacterial↗
PubMed1987

Plasmid transformation of Azotobacter vinelandii OP.

Azotobacter vinelandii OP which had been naturally induced to competence by growth in iron- and molybdenum-limited medium was transformed with the broad-host-range cloning vector pKT210. However, the transformation frequency at nearly saturating levels of DNA was 1000-fold lower for pKT210 than for a single chromosomal DNA marker (nif+). Plasmid- and chromosomal-DNA-mediated transformation events were competitive, magnesium-dependent, 42 degrees C-sensitive processes specific to double-stranded DNA, suggesting a common mechanism of DNA binding and uptake. The low frequency of plasmid transformation was not related to restriction of transforming DNA or to the growth period allowed for phenotypic expression. Covalently-closed-circular and open-circular forms of pKT210 transformed cells equally well whereas EcoRI- or HindIII-linearized pKT210 transformed cells with two to three times greater efficiency. Genetic transformation was enhanced 10- to 50-fold when pKT210 contained an insert fragment of A. vinelandii nif DNA, indicating that A. vinelandii possessed a homology-facilitated transformation system. However, all transformants failed to maintain the plasmid-encoded antibiotic resistance determinants, and extrachromosomal plasmid DNA was not recovered from these cells. Flush-ended pKT210 was not active in transformation; however, competent cells were transformed to Nif+ by HincII-digested plasmid DNA containing the cloned A. vinelandii nif-10 marker.

Azotobacter↗
PubMed1980

Deoxyribonucleic acid-binding properties and membrane protein composition of a competence-deficient mutant of Haemophilus influenzae.

A mutant of Haemophilus influenzae was isolated which was completely unable to take up double-stranded homologous deoxyribonucleic acid (DNA) at normal physiological conditions but which took up DNA equally as well as the wild type at low pH (pH 4.4). The properties of the mutant provide evidence for the existence of two different mechanisms for DNA entry in the H. influenzae transformation system. With the aid of the mutant the optimal conditions for entry of DNA by these two mechanisms were determined, and the dependence of entry and the specific transforming activity of the entered DNA on competence was examined. The mechanism of entry of DNA at neutral pH, which is not functioning in the mutant, effected entry of homologous DNA only, whereas the mechanism involved in entry of DNA at low pH also effected entry of heterologous DNA. This suggests that the mutant is lacking a protein which recognizes the specific base sequence(s) required for entry. Comparison of the protein composition of the membranes of mutant cells subjected to a growth regimen provoking competence in wild-type cells with that of competent wild-type cells revealed that the mutant is impaired in the synthesis of a protein with a molecular weight of 22,500.

Bacterial Proteins↗
PubMed1974

Specificity in deoxyribonucleic acid uptake by transformable Haemophilus influenzae.

Cells of Haemophilus influenzae strain Rd competent for genetic transformation irreversibly bound approximately five molecular fragments of H. influenzae deoxyribonucleic acid (DNA) per cell; under identical conditions, DNA derived from Escherichia coli B was not taken up (<1 molecule per 50 cells). Similarly, DNA from Xenopus laevis was not taken up by competent H. influenzae. Of the heterologous DNAs tested, only DNA from H. parainfluenzae interfered with the uptake of H. influenzae DNA, as judged by competition experiments employing either DNA binding or genetic transformation as the test system. The extracellular heterologous DNA did not suffer either single- or double-strand breakage upon exposure to competent H. influenzae.

Animals↗
PubMed1983

Heat sensitivity of Azotobacter vinelandii genetic transformation.

Heating competent Azotobacter vinelandii at 37 or 42 degrees C resulted in a total loss of competence with no loss of viability. The transformation process was relatively insensitive to heating at either temperature once DNase-resistant DNA binding was nearly complete. Although competent and 42 degrees C-treated cells bound equivalent amounts of [32P]DNA in a DNase-resistant state, no donor DNA marker (nif) or radioactivity was detected in the envelope-free cell lysate of heated cells, suggesting that DNA transport across the cell envelope was a heat-sensitive event. Competence was reacquired in a 42 degrees C-treated culture after 2 h of incubation at 30 degrees C by a process which required RNA and protein syntheses. The release of a surface glycoprotein, required for competence, from cells treated at 42 degrees C occurred in an insufficient amount to account for the total loss of competence. Recovery of competence in 42 degrees C-treated cells and further transformation of competent cells were prevented by the exposure of cells to saturating amounts of transforming DNA. Further DNase-resistant DNA binding, however, still occurred, suggesting that there were two types of receptors for DNase-resistant DNA binding to competent A. vinelandii. DNase-resistant DNA binding was dependent on magnesium ions, and at least one receptor type did not discriminate against heterologous DNA.

Azotobacter↗
PubMed1981

Competence for DNA transfer of ouabain resistance and thymidine kinase: clonal variation in mouse L-cell recipients.

We have used the calcium phosphate precipitation technique to study the competence of mammalian cell recipients for transformation with genomic mammalian cell DNA. The transformation efficiency for thymidine kinase (tk) varies 10- to 20-fold (up to 10(-4) transformants/recipient) among different subclones of the LM tk- CL 1D mouse fibroblast cell line. Analysis of this phenotype among second-generation subclones indicates that subclones exhibiting high competence tend to breed true, whereas those with low competence do not. Isolation of Tk- revertants from TK+ transformants results in the selection of cells with a high-competence phenotype as measured by their subsequent transformation for tk. This phenotype appears to be a general characteristic of such cells because recipients more competent for transfer of a second marker, ouabain resistance (ouaR). This codominant marker coding for the Na K+-ATPase can be transferred at frequencies of 10(-5) in the high-competence recipients. These results indicate that competence for DNA-mediated gene transfer can be determined in part by genetic factors.

Animals↗
PubMed1994

Segregation properties of bovine papillomaviral plasmid DNA.

Essentially complete segregation of replication-competent BPV-1 plasmid DNA species was observed in daughter subclones derived from primary co-transformed C127 cell lines. Thus, whereas primary co-transformants retained both of two distinguishable co-transfected plasmid species, subcloning experiments revealed that morphologically transformed daughter subclones derived from such co-transformed cell lines contained only one species of viral plasmid DNA. Similar results were obtained with each of two conveniently marked replication and transformation-competent mutants: one with a linker-insertion in the viral upstream regulatory region, and one with a 260 base-pair deletion within the L2 (late) gene, which has no recognized role in plasmid replication or stability. Morphological revertant cell clones that contained no detectable viral plasmid DNA genomes were also isolated at a surprisingly high frequency from clonal wild-type BPV-1 transformed cell lines and from cell lines transformed by various BPV-1 mutants. Further co-transfection experiments were done with a combination of transformation-competent and transformation-defective BPV-1 genomes to investigate a possible role for a viral oncogene in plasmid persistence. In this case, elimination of the transformation-defective mutant was observed after the initial establishment of both input genomes as replicating plasmids in cell clones morphologically transformed by the transformation-competent viral mutant with an intact E5 oncogene. No cell subclones were isolated that contained only the transformation-defective mutant, implying that it was defective in long-term plasmid persistence. Our results indicate that there is significant randomization in the processes of replication and/or partitioning of the BPV-1 genome in mouse C127 cells, and, in combination with previous observations, also suggest that BPV plasmid persistence in C127 cell lines may be the result of a selective proliferative advantage conferred on virus-infected cells by viral oncogene-induced cell growth transformation.

Animals↗
PubMed2000

Survival of free DNA encoding antibiotic resistance from transgenic maize and the transformation activity of DNA in ovine saliva, ovine rumen fluid and silage effluent.

To assess the likelihood that the bla gene present in a transgenic maize line may transfer from plant material to the microflora associated with animal feeds, we have examined the survival of free DNA in maize silage effluent, ovine rumen fluid and ovine saliva. Plasmid DNA that had previously been exposed to freshly sampled ovine saliva was capable of transforming competent Escherichia coli cells to ampicillin resistance even after 24 h, implying that DNA released from the diet could provide a source of transforming DNA in the oral cavity of sheep. Although target DNA sequences could be amplified by polymerase chain reaction from plasmid DNA after a 30-min incubation in silage effluent and rumen contents, only short term biological activity, lasting less than 1 min, was observed in these environments, as shown by transformation to antibiotic resistance. These experiments were performed under in vitro conditions; therefore further studies are needed to elucidate the biological significance of free DNA in the rumen and oral cavities of sheep and in silage effluent.

Animals↗
PubMed2003

Genome plasticity in Acinetobacter: new degradative capabilities acquired by the spontaneous amplification of large chromosomal segments.

In Acinetobacter sp. ADP1, growth on benzoate requires regulation of the cat genes by two transcriptional activators. Here, mutants were obtained from a strain lacking both activators by selecting for growth on benzoate medium. The mutants, which arose within 3 weeks at a frequency of approximately 10-8, carried amplified chromosomal regions (amplicons) encompassing the cat genes. Multiple occurrences of low-level expression of catA and the catBCIJFD operon provided sufficient transcription for growth. The amplicons of four independently isolated mutants varied in size from approximately 30-100 kbp of the normally 3.8 Mbp chromosome. Mutants had approximately 10-20 copies of an amplicon in adjacent head-to-tail orientations. At the amplicon's chromosomal endpoint, an atypical junction juxtaposed normally distant DNA regions from opposite sides of the cat genes. The sequences of these junctions revealed the precise recombination sites underlying amplification. Additionally, amplicon stability was evaluated in the absence of selective pressure. The natural competence of Acinetobacter for transformation by linear DNA has allowed the development of a powerful new model system for investigating chromosomal rearrangements and for engineering DNA amplifications for wide-ranging applications. The frequent spontaneous amplification of these large chromosomal segments demonstrated the importance of supra-operonic gene clustering in the evolution of catabolic pathways.

Acinetobacter↗
PubMed1999

Reversible gels for electrophoresis and isolation of DNA.

Here, the application of the gel-forming carbohydrate polymer, gellan gum, for the electrophoresis and isolation of DNA is detailed. Gellan gun forms gels in the presence of divalent metal cations, and the gels can be converted back to a solution by the addition of a chelating agent such as EDTA. Also, gellan electrophoresis gels can be formed using diamines. These gels are reversible by increasing the pH, which results in the deprotonation of the diamine. Gellan electrophoresis gels were used for separations at concentrations as low as 0.03%. Native gellan electrophoresis gels have significant electroosmosis and were generally run overnight. A gellan electrophoresis gel (0.1%) showed good resolution of DNA from approximately 50-1 kbp. The addition of linear polymers, such as hydroxethyl cellulose, to the gellan gum before casting greatly reduced the electroosmosis. The additional polymer increased the resolution of low-molecular-weight DNA down to approximately 200 bp and allowed gels to be run in a few hours. DNA isolated from gellan electrophoresis gels could be cut by common restriction enzymes and ligated in the presence of the gellan gum. The presence of gellan gum did not significantly inhibit the transformation of competent E. coli cells by plasmid DNA.

Biotechnology↗
PubMed2005

Co-ordinated bacteriocin production and competence development: a possible mechanism for taking up DNA from neighbouring species.

It is important to ensure DNA availability when bacterial cells develop competence. Previous studies in Streptococcus pneumoniae demonstrated that the competence-stimulating peptide (CSP) induced autolysin production and cell lysis of its own non-competent cells, suggesting a possible active mechanism to secure a homologous DNA pool for uptake and recombination. In this study, we found that in Streptococcus mutans CSP induced co-ordinated expression of competence and mutacin production genes. This mutacin (mutacin IV) is a non-lantibiotic bacteriocin which kills closely related Streptococcal species such as S. gordonii. In mixed cultures of S. mutans and S. gordonii harbouring a shuttle plasmid, plasmid DNA transfer from S. gordonii to S. mutans was observed in a CSP and mutacin IV-dependent manner. Further analysis demonstrated an increased DNA release from S. gordonii upon addition of the partially purified mutacin IV extract. On the basis of these findings, we propose that Streptococcus mutans, which resides in a multispecies oral biofilm, may utilize the competence-induced bacteriocin production to acquire transforming DNA from other species living in the same ecological niche. This hypothesis is also consistent with a well-known phenomenon that a large genomic diversity exists among different S. mutans strains. This diversity may have resulted from extensive horizontal gene transfer.

Bacterial Proteins↗
PubMed2004

An inverse PCR technique to rapidly isolate the flanking DNA of dictyostelium insertion mutants.

Restriction enzyme mediated integration is a widely used and effective method for insertional mutagenesis in Dictyostelium discoideum. In this method, plasmid rescue is used to clone the genomic deoxyribonucleic acid (DNA) sequences that flank the insertion site. For this to be effective, it is necessary to first find a convenient restriction enzyme site within the genomic DNA. This is a time-consuming process that requires Southern blot analysis of the mutant DNA. In addition, plasmid rescue requires transformation into highly competent Escherichia coli. Problems can arise owing to unstable genomic sequences, damage to the plasmid DNA and exogenous plasmid contamination. We have established a simple and rapid polymerase chain reaction-based technique that works for all mutants and circumvents the need for Southern blot analysis and plasmid rescue.

Animals↗
PubMed1994

The Haemophilus influenzae sxy-1 mutation is in a newly identified gene essential for competence.

The sxy-1 mutation of Haemophilus influenzae causes a 100- to 1,000-fold increase in spontaneous natural competence. We have used mapping and sequencing to identify this mutation as a G-to-A transition in an open reading frame adjacent to the rec-1 locus. This mutation substitutes valine for isoleucine at amino acid 19 of the protein specified by this gene (now named sxy). A multicopy plasmid containing the wild-type sxy gene confers constitutive competence on wild-type cells. Cells carrying this plasmid exhibit, in all stages of growth, DNA uptake levels and transformation frequencies as high those normally seen only after full induction of competence by starvation; deletion of part of the sxy gene from the plasmid abolishes this effect. In contrast, a transposon insertion in sxy entirely prevents both DNA uptake and transformation, indicating that sxy encodes a function essential for competence. These findings suggest that sxy may act as a positive regulator of competence. However, because cells carrying the transposon-inactivated sxy::Tn allele grow slowly under conditions that do not induce competence, sxy may also have a role in noncompetent cells.

Alleles↗
PubMed2026

Unveiling a missing component of the atypical type IV secretion system required for natural transformation of Helicobacter pylori.

Exchange of genetic information by natural transformation shapes bacterial evolution. In Helicobacter pylori it is thought to drive its unusually high recombination rate, which has a crucial role in the evolution of virulence and the propagation of antibiotics resistance genes. While in most cases uptake of the incoming DNA into the periplasm is mediated by type IV pili, in H. pylori this initial step of natural transformation requires ComB, a unique competence-specific type IV secretion system (T4SS). The mechanisms by which ComB mediates DNA uptake are still poorly understood, since T4SS are usually involved in an opposite process of DNA export. Here, we identify a gene (hp1421) that is absolutely required for uptake of the transforming DNA into the periplasm, although distant from the comB operons. We show that hp1421 codes for a hexameric ATPase from the VirB11 family. HP1421 is present in the cytoplasm and interacts with ComB4, another ATPase of the T4SS inner membrane subcomplex. The structural modelling and functional analysis of HP1421 and its interaction with ComB4 indicate that HP1421 is a missing component of the ComB inner-membrane subcomplex that we propose to name ComB11. Phylogenetic analyses show that comB11 is a H. pylori core gene and suggest that the competence-dedicated ComB T4SS was a recent acquisition within Helicobacteraceae. Hence, co-option of the T4SS for DNA transformation requires nearly all the proteins that were previously essential for DNA conjugation.

Helicobacter pylori↗
PubMed2026

Unveiling a missing component of the atypical type IV secretion system required for natural transformation of Helicobacter pylori.

Exchange of genetic information by natural transformation shapes bacterial evolution. In Helicobacter pylori it is thought to drive its unusually high recombination rate, which has a crucial role in the evolution of virulence and the propagation of antibiotics resistance genes. While in most cases uptake of the incoming DNA into the periplasm is mediated by type IV pili, in H. pylori this initial step of natural transformation requires ComB, a unique competence-specific type IV secretion system (T4SS). The mechanisms by which ComB mediates DNA uptake are still poorly understood, since T4SS are usually involved in an opposite process of DNA export. Here, we identify a gene (hp1421) that is absolutely required for uptake of the transforming DNA into the periplasm, although distant from the comB operons. We show that hp1421 codes for a hexameric ATPase from the VirB11 family. HP1421 is present in the cytoplasm and interacts with ComB4, another ATPase of the T4SS inner membrane subcomplex. The structural modelling and functional analysis of HP1421 and its interaction with ComB4 indicate that HP1421 is a missing component of the ComB inner-membrane subcomplex that we propose to name ComB11. Phylogenetic analyses show that comB11 is a H. pylori core gene and suggest that the competence-dedicated ComB T4SS was a recent acquisition within Helicobacteraceae. Hence, co-option of the T4SS for DNA transformation requires nearly all the proteins that were previously essential for DNA conjugation.

Journal Article↗
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↗
PubMed1987

Competence for genetic transformation in Streptococcus pneumoniae: molecular cloning of com, a competence control locus.

To identify and map genes involved in competence for genetic transformation in Streptococcus pneumoniae, we have cloned DNA surrounding an ermB insertion mutation that causes a competence factor deficiency. We recovered the insert and approximately 500 base pairs of neighboring pneumococcal DNA in pMB9. Larger pieces of DNA from this region were unstable in pMB9 and pBR325. However, larger pieces were stable in pKK232-8, an Escherichia coli vector containing strong transcription terminators. Overlapping pieces of wild-type DNA from this competence control region were cloned and mapped in this vector. Insertion mutations were constructed in vitro throughout the cloned region. When crossed into the pneumococcus chromosome, they showed that the com locus was 4.2 to 5.2 kilobases long.

Bacterial Proteins↗