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

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

Transpososomes: stable protein-DNA complexes involved in the in vitro transposition of bacteriophage Mu DNA

We report that two types of stable protein-DNA complexes, or transpososomes, are generated in vitro during the Mu DNA strand transfer reaction. The Type 1 complex is an intermediate in the reaction. Its formation requires a supercoiled mini-Mu donor plasmid, Mu A and HU protein, and Mg2+. In the Type 1 complex the two ends of Mu are held together, creating a figure eight-shaped molecule with two independent topological domains; the Mu sequences remain supercoiled while the vector DNA is relaxed because of nicking. In the presence of Mu B protein, ATP, target DNA, and Mg2+, the Type 1 complex is converted into the protein-associated product of the strand transfer reaction. In this Type 2 complex, the target DNA has been joined to the Mu DNA ends held in the synaptic complex at the center of the figure eight. Supercoils are not required for the latter reaction.     

Steady-state kinetic analysis of ATP hydrolysis by the B protein of bacteriophage mu. Involvement of protein oligomerization in the ATPase cycle

The DNA strand-transfer reaction of bacteriophage Mu requires Mu B protein and ATP for high efficiency. These factors facilitate the capture of target DNA by the donor protein-DNA complex. To understand the mechanism of the Mu B ATPase cycle in the Mu DNA strand-transfer reaction, we undertook a steady-state kinetic analysis of Mu B ATPase. The results reveal complex properties of the ATPase activity; Mu B protein oligomerizes in the presence of ATP, and ATP hydrolysis by the Mu B ATPase is stimulated by the protein oligomerization and shows a positive cooperativity with respect to ATP concentration. Mu B ATPase activity is also modulated by DNA and Mu A protein. DNA alone suppresses the catalytic activity of Mu B ATPase, whereas DNA enhances the apparent binding affinity for ATP. In the presence of Mu A protein together with DNA, however, the catalytic activity is greatly stimulated. Based on these results, we propose a working hypothesis in which oligomerization of Mu B protein plays a key role in its ATPase cycle.     

Fate of heterospecific transforming DNA bound to Streptococcus sanguis.

The fate of 3H-labeled str-r fus-s DNA from Streptococcus pneumoniae, bound after a 1-min uptake to 14C-labeled str-s fus-r S. sanguis recipients, was followed by techniques previously developed for analyzing the fate of homospecific DNA. Heterospecific S. pneumoniae DNA was bound and formed complexes with recipient protein in a manner similar to that of homospecific DNA but transformed relatively poorly. The rate at which complexed heterospecific DNA becomes physically associated with recipient DNA, and at which donor markers are integrated into the chromosome, was slower than in the case of homospecific DNA. In addition, about half of the heterospecific donor counts initially bound in trichloracetic acid-insoluble form were gradually solubilized and released from the cell. The association of heterospecific DNA with the recipient chromosome was more unstable than that involving homospecific DNA, since only associations of the former type were largely dissociated by isolation and resedimentation. The donor DNA-containing material so dissociated had the same sedimentation properties as complexed heterospecific DNA before association, indicating that the complex of single-stranded donor DNA and recipient protein formed on uptake moves as a whole from its site of formation to synapse with the chromosome.     

Type IV secretion: the Agrobacterium VirB/D4 and related conjugation systems

The translocation of DNA across biological membranes is an essential process for many living organisms. In bacteria, type IV secretion systems (T4SS) are used to deliver DNA as well as protein substrates from donor to target cells. The T4SS are structurally complex machines assembled from a dozen or more membrane proteins in response to environmental signals. In Gram-negative bacteria, the conjugation machines are composed of a cell envelope-spanning secretion channel and an extracellular pilus. These dynamic structures (i) direct formation of stable contacts-the mating junction-between donor and recipient cell membranes, (ii) transmit single-stranded DNA as a nucleoprotein particle, as well as protein substrates, across donor and recipient cell membranes, and (iii) mediate disassembly of the mating junction following substrate transfer. This review summarizes recent progress in our understanding of the mechanistic details of DNA trafficking with a focus on the paradigmatic Agrobacterium tumefaciens VirB/D4 T4SS and related conjugation systems.     

Transposition of Mu DNA: joining of Mu to target DNA can be uncoupled from cleavage at the ends of Mu

Transposition of Mu involves transfer of the 3' ends of Mu DNA to the 5' ends of a staggered cut in the target DNA. We find that cleavage at the 3' ends of Mu DNA precedes cutting of the target DNA. The resulting nicked species exists as a noncovalent nucleoprotein complex in which the two Mu ends are held together. This cleaved donor complex completes strand transfer when a target DNA, Mu B protein, and ATP are provided. Mu end DNA sequences that have been precisely cut at their 3' ends by a restriction endonuclease, instead of by Mu A protein and HU, are efficiently transferred to a target DNA upon subsequent incubation with Mu A protein, Mu B protein, and ATP. Cleavage of the Mu ends therefore cannot be energetically coupled with joining these ends to a target DNA. We discuss the DNA strand transfer mechanism in view of these results, and propose a model involving direct transfer of the 5' ends of the cut target DNA, from their original partners, to the 3' ends of Mu.     

Heterospecific transformation in Bacillus subtilis: protein composition of a membrane-DNA complex containing unstable heterologous donor-recipient complex

Summary Previously it was demonstrated that, in contrast to the homologous donor-recipient complex, the unstable heterologous donor-recipient complex remains bound to the cellular membrane. To examine whether proteins known to be involved in the processing of transforming DNA in Bacillus subtilis are associated with membrane fragments which carry chromosomal DNA, a crude membrane-DNA complex was subjected to electrophoresis through a sucrose gradient. This resulted in the separation of membrane fragments associated with DNA and free membrane fragments. By means of two-dimensional gel electrophoresis several proteins, either uniquely present or considerably enriched in the purified membrane-DNA complex, were detected. Among these proteins we identified the 45 kD recE gene product, required for recombination, the 18 kD binding protein involved in the binding of transforming DNA and a 17 kD nuclease involved in the entry of transforming DNA.These results suggest that the membrane sites at which donor DNA integrates into the recipient chromosome are in the vicinity of the sites of entry of donor DNA through the membrane.Abbreviations DNAase I deoxyribonuclease I - DRC donor-recipient DNA complex - PEG polyethyleneglycol - PMSF phenylmethylsulphonylfluoride - SSC standard saline citrate - TCA trichloroacetic acid     

Inversion of the phosphate chirality at the target site of Mu DNA strand transfer: evidence for a one-step transesterification mechanism.

Central to transposition of phage Mu are two reactions mediated by the MuA protein. First, MuA introduces single-stranded cuts at the ends of the Mu DNA to generate 3' OH termini. In the subsequent strand-transfer step, the MuA-Mu DNA end complex cuts a target DNA and joins the Mu 3' ends to the 5' ends of the target. DNA containing chiral phosphorothioates was used to demonstrate inversion of the chirality during the course of strand transfer. This result strongly supports a one-step transesterification mechanism in which the 3' OH of the cleaved donor DNA is the attacking nucleophile. Furthermore, this donor 3' OH group was essential for target DNA cleavage. In contrast, during lambda integration the phosphate chirality was retained, as expected for a two-step transesterification involving a covalent protein-DNA intermediate.     

Fate of Transforming Deoxyribonucleate in Bacillus subtilis

The majority of donor deoxyribonucleate (DNA) at early stages after uptake was found in a complex with a cell component which changes its buoyant behavior on equilibrium density gradients. Analysis of the recipient cell lysates, after treatment to dissociate the complex, showed about two-thirds of the donor molecules in denatured form and the rest associated with recipient DNA. Incubation of cells after DNA uptake leads to the disappearance of denatured donor DNA and to the increase of donor label associated with recipient DNA. Some characteristics of a component from intact cells or spheroplasts with affinity for denatured Bacillus subtilis DNA are described.     

Transformation in pneumococcus: protein content of eclipse complex.

A two-step purification of pneumococcal eclipse complex is described, which uses sucrose gradient sedimentation followed by agarose gel permeation chromatography. Purified complex contains, in addition to donor DNA single strands, macromolecular material that can be labeled with methionine or leucine during development of competence. This material co-chromatographed with eclipse complex DNA on hydroxylapatite, was dissociated from the DNA by sodium dodecyl sulfate, and was completely digested by Pronase. The sodium dodecyl sulfate-released material eluted as a single peak in sodium dodecyl sulfate chromatography. These properties were consistent with the noncovalent association with eclipse complex of a protein or class of proteins with a narrow range of polypeptide sizes. Evidence for the specific association of this protein with transforming DNA is eclipse was also obtained from parallel purification from 35S-labeled nontransformed cells; the amount of methionine label in the corresponding fractions in such cells was only 5% of that in transformed cells.     

An unstable donor-recipient DNA complex in transformation of Bacillus subtilis.

Summary In re-extracted DNA obtained shortly after uptake of transforming DNA by Bacillus subtilis, increased amounts of donor DNA radioactivity banding at the position of donor-recipient DNA complex (DRC) are observed in CsCl gradients, if the cells are irradiated with high doses of UV prior to reextraction of the DNA. Qualitatively, the same phenomenon is observed if lysates of transforming cells are irradiated. UV-irradiation of lysates of competent cells to which single-stranded DNA is added after lysis, does not result in linkage of this DNA to the chromosomal DNA. Two observations argue in favour of the formation of a specific labile complex between donor and resident DNA during transformation. Firstly, heterologous donor DNA from Escherichia coli, although being processed to single-stranded DNA in competent B. subtilis, does not seem to be linked to the recipient chromosome upon UV-irradiation, and secondly, the labile complex of donor and recipient DNA can be stabilized by means of treatment of the lysates of transforming cells with 4, 5¹, 8-trimethylpsoralen in conjuction with long-wave ultra violet light irradiation. This indicates that base-pairing is involved in the formation of the complex. On the basis of these results we assume that the unstable complex of donor and recipient DNA is an early intermediate in genetic recombination during transformation.     

Dynamics of synaptic SfiI-DNA complex: single-molecule fluorescence analysis

A single-molecule analysis was applied to study the dynamics of synaptic and presynaptic DNA-protein complexes (binding of two DNA and one DNA duplex, respectively). In the approach used in this study, the protein was tethered to a surface, allowing a freely diffusing fluorescently labeled DNA to bind to the protein, thus forming a presynaptic complex. The duration of fluorescence burst is the measure of the characteristic lifetime of the complex. To study the formation of the synaptic complex, the two SfiI-bound duplexes with the labeled donor and acceptor were used. The synaptic complex formation by these duplexes was detected by the fluorescence resonance energy transfer approach. The duration of the fluorescence resonance energy transfer burst is the measure of the characteristic lifetime of the synaptic complex. We showed that both synaptic and presynaptic complexes have characteristic dissociation times in the range of milliseconds, with the synaptic SfiI-DNA complex having the shorter dissociation time. Comparison of the off-rate data for the synaptic complex with the rate of DNA cleavage led to the hypothesis that the complex is very dynamic, so the formation of an enzymatically active synaptic complex is a rather rare event in these series of conformational transitions.     

Transformation in Streptococcus pneumoniae: formation of eclipse complex in a coiA mutant implicates CoiA in genetic recombination

CoiA is a transient protein expressed specifically during competence and required for genetic transformation in Streptococcus pneumoniae, but not for DNA uptake. It is widely conserved among Gram-positive bacteria but its function is unknown. Here we report that although the rate of DNA uptake was not affected in a coiA mutant, the internalized donor DNA did not recombine into the host chromosome to form a physical and genetic heteroduplex. Instead, DNA taken up by a coiA mutant accumulated in the form of a single-stranded (ss) DNA-protein complex indistinguishable from the eclipse complex formed as a recombination intermediate in wild-type competent cells. Internalized donor DNA in a dprA mutant did not accumulate either as ss DNA or as an eclipse complex. Together, these results establish that a coiA mutant exhibits a phenotype different from that of dprA or recA mutants, and that CoiA functions at a later step in promoting recombination during genetic transformation in Streptococcus pneumoniae.     

Fate of transforming DNA following uptake by competent Bacillus subtilis. I. Formation and properties of the donor-recipient complex

Following uptake by competent Bacillus subtilis, transforming DNA is converted to two distinct slowly sedimenting molecular forms which possess little transforming activity (eclipse). A few minutes after uptake is initiated, a physical complex of donor and recipient DNA begins to form. The recovery of donor transforming activity following eclipse, and the appearance of recombinant activity, previously reported by Venema, Pritchard &; Venema-Schröder (1965), is shown to be due to changes occurring in the donor—recipient complex. This complex exists transiently in a form with low recombinant-type transforming activity. This transient form may be one in which the donor and recipient components are joined non-covalently. The donor-recipient complex is shown to be a heteroduplex structure in which the donor moiety has an approximate molecular weight of 750,000.     

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.     

Role of DNA topology in Mu transposition: mechanism of sensing the relative orientation of two DNA segments

DNA strand transfer at the initiation of Mu transposition normally requires a negatively supercoiled transposon donor molecule, containing both ends of Mu in inverted repeat orientation. We propose that the specific relative orientation of the Mu ends is needed only to energetically favor a particular configuration that the ends must adopt in a synaptic complex. The model was tested by constructing special donor DNA substrates that, because of their catenation or knotting, energetically favor this same configuration of the Mu ends, even though they are on separate molecules or in direct repeat orientation. These structures are efficient substrates for the strand transfer reaction, whereas appropriate control structures are not. The result eliminates tracking or protein scaffold models for orientation preference. Several other systems in which the relative orientation of two DNA segments is sensed may utilize the same mechanism.     

Transformation in Bacillus subtilis: involvement of the 17-kilodalton DNA-entry nuclease and the competence-specific 18-kilodalton protein.

A protein complex, consisting of a 17-kilodalton (kDa) nuclease and an 18-kDa protein, is believed to be involved in the binding and entry of donor DNA during transformation of Bacillus subtilis (H. Smith, K. Wiersman, S. Bron, and G. Venema, J. Bacteriol. 156:101-108, 1983). In this paper, the nucleotide sequences of the genes encoding both the nuclease and the 18-kDa protein are presented. The genes are encoded by a 904-base-pair PstI-HindIII fragment. The open reading frames encoding both proteins are partly overlapping. A B. subtilis mutant was constructed by insertion of a Cmr marker into the gene encoding the nuclease. This mutant lacked the competence-specific nuclease activity and the 18-kDa protein but retained 5% residual transformation. The total DNA association of the mutant was higher than that of the wild-type cells, and DNA entry was reduced to 30% of the wild-type level. These results suggest that an alternative pathway exists for the internalization of transforming DNA. A mutant, exclusively deficient for the 18-kDa protein, previously suggested to be involved in the binding of transforming DNA, was constructed by insertion of a kanamycin resistance gene into the coding sequence of the gene. Since the mutant showed wild-type DNA-binding activity, the 18-kDa protein is probably not involved in the binding of donor DNA to competent cells. The transforming activity of the mutant was reduced to 25% of the wild-type level, indicating that the 18-kDa protein has a function in the transformation process. In vitro experiments showed that the 18-kDa protein is capable of inhibiting the activity of the competence-specific nuclease. Its possible role in transformation is discussed.     

Translocation of the pre-synaptic complex formed upon DNA uptake by Streptococcus sanguis and its inhibition by ethidium bromide.

Summary Donor DNA in its initially bound, singlestranded form exists in a chromosomally-unassociated complex where it is resistant to exogenous DNase I but sensitive to micrococcal nuclease. Most of the complexes are readily recuperable from the supernatant of recipients converted into spheroplasts. Subsequent to formation of this superficially located complex, donor DNA progressively associates with the recipient chromosome into which it is eventually integrated. Treatment of recipients with ethidium bromide at various times after initial DNA binding almost immediately halts translocation of whatever donor material is not yet synapsed with the chromosome. On the other hand, donor DNA that has already synapsed experiences no difficulty in becoming genetically integrated. Some degradation occurs to DNA that fails to undergo translocation as a result of ethidium bromide treatment, the acid-soluble products appearing in the culture medium. DNA in untranslocated complexes surviving treatment is not appreciably different in single-strand length from that in untreated complexes. When these surviving complexes are isolated from a cell lysate, the contained DNA can be shown by spectrofluorometry to have bound the drug.     

Transformation in Bacillus subtilis: characterization of an intermediate in recombination observed during transformation.

Summary From recombination-proficient competent cells of Bacillus subtilis in which the donor DNA entered at 17°, and which were kept at the same temperature, a complex of donor DNA and the recipient chromosome can be obtained which has a relatively high buoyant density in CsCl gradients. Exposure of the isolated complex to nuclease S1 liberates donor radioactivity. The limited biological activity of DNA re-extracted from cells attempting to recombine at 17° is decreased upon incubation with nuclease S1. If recombination is allowed to proceed at 30°, the high buoyant density of the donor-recipient complex decreases to normal values and less radioactivity can be liberated from the complex by nuclease S1. Concomitantly the biological activity of re-extracted DNA becomes less vulnerable to nuclease S1 under these conditions. On the basis of these observations we assume that the intermediate complex partly consists of unpaired single-stranded donor DNA.Support for the correctness of this assumption is derived from experiments with a mutant, which is delayed in the processing of high buoyant density donor-recipient complex to normal buoyant density donor-recipient complex. This delay is reflected in the time of acquisition of resistance to nuclease S1 digestion of the isolated complex.     

Fate of homospecific transforming DNA bound to Streptococcus sanguis.

The fate of [3H]DNA from Streptococcus sanguis str-r43 fus-s donors in [14C]S. sanguis str-s fus-r1 recipients was studied by examining the lysates prepared from such recipients at various times after 1 min of exposure to DNA. The lysates were analyzed in CsCl and 10 to 30% sucrose gradients; fractions from the gradients were tested for biological activity and sensitivity to nucleases, subjected to various treatments and retested for nuclease sensitivity, and run on 5 to 20% neutral and alkaline sucrose gradients. The results demonstrate that donor DNA bound to S. sanguis cells in a form resistant to exogenous deoxyribonuclease is initially single stranded and complexed to recipient material. Donor DNA can be removed from the complex upon treatment of the complex with Pronase, phenol, or isoamyl alcohol-chloroform. Within the complex, donor DNA is relatively insensitive to S1 endonuclease but can regain its sensitivity by treatment with phenol. With time the complex moves as a whole to associate physically with the recipient chromosome. After a noncovalent stage of synapsis, donor material is covalently bonded to and acquires the nuclease sensitivity of recipient DNA, while donor markers regain transforming activity and become linked to resident markers.