Flavones inhibit the hexameric replicative helicase RepA.

Helicases couple the hydrolysis of nucleoside triphosphates (NTPs) to the unwinding of double-stranded nucleic acids and are essential in DNA metabolism. Thus far, no inhibitors are known for helicases except heliquinomycin isolated from Streptomyces sp. As the three-dimensional structure of the hexameric replicative DNA helicase RepA encoded by the broad host-range plasmid RSF1010 is known, this protein served as a model helicase to search for inhibitory compounds. The commercially available flavone derivatives luteolin, morin, myricetin and dimyricetin (an oxidation product of myricetin) inhibited the ATPase and double-stranded DNA unwinding activities of RepA. Dimyricetin was the most effective inhibitor for both activities. Single-stranded DNA-dependent RepA ATPase activity is inhibited non-competitively by all four compounds. This finding contrasts the inhibition of phosphoinositide 3-kinase by flavones that fit into the ATP binding pocket of this enzyme. Myricetin also inhibited the growth of a Gram-positive and a Gram-negative bacterial species. As we found other hexameric and non-hexameric prokaryotic helicases to be differentially sensitive to myricetin, flavones may provide substructures for the design of molecules helpful for unraveling the mechanism of helicase action and of novel pharmacologically useful molecules.

The genetic organization and evolution of the broad host range mercury resistance plasmid pSB102 isolated from a microbial population residing in the rhizosphere of alfalfa.

Employing the biparental exogenous plasmid isolation method, conjugative plasmids conferring mercury resistance were isolated from the microbial community of the rhizosphere of field grown alfalfa plants. Five different plasmids were identified, designated pSB101-pSB105. One of the plasmids, pSB102, displayed broad host range (bhr) properties for plasmid replication and transfer unrelated to the known incompatibility (Inc) groups of bhr plasmids IncP-1, IncW, IncN and IncA/C. Nucleotide sequence analysis of plasmid pSB102 revealed a size of 55 578 bp. The transfer region of pSB102 was predicted on the basis of sequence similarity to those of other plasmids and included a putative mating pair formation apparatus most closely related to the type IV secretion system encoded on the chromosome of the mammalian pathogen Brucella sp. The region encoding replication and maintenance functions comprised genes exhibiting different degrees of similarity to RepA, KorA, IncC and KorB of bhr plasmids pSa (IncW), pM3 (IncP-9), R751 (IncP-1beta) and RK2 (IncP-1alpha), respectively. The mercury resistance determinants were located on a transposable element of the Tn5053 family designated Tn5718. No putative functions could be assigned to a quarter of the coding capacity of pSB102 on the basis of comparisons with database entries. The genetic organization of the pSB102 transfer region revealed striking similarities to plasmid pXF51 of the plant pathogen Xylella fastidiosa.

Maturation of IncP pilin precursors resembles the catalytic Dyad-like mechanism of leader peptidases.

The pilus subunit, the pilin, of conjugative IncP pili is encoded by the trbC gene. IncP pilin is composed of 78 amino acids forming a ring structure (R. Eisenbrandt, M. Kalkum, E.-M. Lai, C. I. Kado, and E. Lanka, J. Biol. Chem. 274:22548-22555, 1999). Three enzymes are involved in maturation of the pilin: LepB of Escherichia coli for signal peptide removal and a yet-unidentified protease for removal of 27 C-terminal residues. Both enzymes are chromosome encoded. Finally, the inner membrane-associated IncP TraF replaces a four-amino-acid C-terminal peptide with the truncated N terminus, yielding the cyclic polypeptide. We refer to the latter process as "prepilin cyclization." We have used site-directed mutagenesis of trbC and traF to unravel the pilin maturation process. Each of the mutants was analyzed for its phenotypes of prepilin cyclization, pilus formation, donor-specific phage adsorption, and conjugative DNA transfer abilities. Effective prepilin cyclization was determined by matrix-assisted laser desorption-ionization-mass spectrometry using an optimized sample preparation technique of whole cells and trans-3-indolyl acrylic acid as a matrix. We found that several amino acid exchanges in the TrbC core sequence allow prepilin cyclization but disable the succeeding pilus assembly. We propose a mechanism explaining how the signal peptidase homologue TraF attacks a C-terminal section of the TrbC core sequence via an activated serine residue. Rather than cleaving and releasing hydrolyzed peptides, TraF presumably reacts as a peptidyl transferase, involving the N terminus of TrbC in the aminolysis of a postulated TraF-acetyl-TrbC intermediate. Under formal loss of a C-terminal tetrapeptide, a new peptide bond is formed in a concerted action, connecting serine 37 with glycine 114 of TrbC.

The protelomerase of temperate Escherichia coli phage N15 has cleaving-joining activity.

Escherichia coli phage N15 encodes the slightly acidic, 630-residue protein of 72.2 kDa called protelomerase (TelN). TelN is a component of the N15 replication system proposed to be involved in the generation of the linear prophage DNA. This linear DNA molecule has covalently closed ends. The reaction converting circular plasmids into linear molecules was catalyzed in vitro. We demonstrate that the product of telN functions as the protelomerase in the absence of other N15-encoded factors. Purified TelN processes circular and linear plasmid DNA containing the proposed target site telRL to produce linear double-stranded DNA with covalently closed ends. The 56-bp telRL target site consists of a central telO palindrome of 22 bp and two 14-bp flanking sequences comprising inverted repeats. telO is separated from these repeats by 3 bp on each side. The telRL sequence is sufficient for TelN-mediated processing. The ends of the DNA molecules generated in vitro have the same configuration as do those observed in vivo. TelN exerts its activity as cleaving-joining enzyme in a concerted action.

Conjugative junctions in RP4-mediated mating of Escherichia coli.

The physical association of bacteria during conjugation mediated by the IncPalpha plasmid RP4 was investigated. Escherichia coli mating aggregates prepared on semisolid medium were ultrarapidly frozen using copper block freezing, followed by freeze substitution, thin sectioning, and transmission electron microscopy. In matings where the donor bacteria contained conjugative plasmids, distinctive junctions were observed between the outer membranes of the aggregates of mating cells. An electron-dense layer linked the stiffly parallel outer membranes in the junction zone, but there were no cytoplasmic bridges nor apparent breaks in the cell walls or membranes. In control experiments where the donors lacked conjugative plasmids, junctions were not observed. Previous studies have shown that plasmid RP4 carries operons for both plasmid DNA processing (Tra1) and mating pair formation (Tra2). In matings where donor strains carried Tra2 only or Tra2 plus the pilin-processing protease TraF, junctions were found but they were shorter and more interrupted than the wild type. If the donor strain had the pilin gene knocked out (trbC), junctions were still found. Thus, it appears that the electron-dense layer between the outer membranes of the conjugating cells is not composed of pilin.

Enzymology of type IV macromolecule secretion systems: the conjugative transfer regions of plasmids RP4 and R388 and the cag pathogenicity island of Helicobacter pylori encode structurally and functionally related nucleoside triphosphate hydrolases.

Type IV secretion systems direct transport of protein or nucleoprotein complexes across the cell envelopes of prokaryotic donor and eukaryotic or prokaryotic recipient cells. The process is mediated by a membrane-spanning multiprotein assembly. Potential NTPases belonging to the VirB11 family are an essential part of the membrane-spanning complex. Three representatives of these NTPases originating from the conjugative transfer regions of plasmids RP4 (TrbB) and R388 (TrwD) and from the cag pathogenicity island of Helicobacter pylori (HP0525) were overproduced and purified in native form. The proteins display NTPase activity with distinct substrate specificities in vitro. TrbB shows its highest specific hydrolase activity with dATP, and the preferred substrate for HP0525 is ATP. Analysis of defined TrbB mutations altered in motifs conserved within the VirB11 protein family shows that there is a correlation between the loss or reduction of NTPase activity and transfer frequency. Tryptophan fluorescence spectroscopy of TrbB and HP0525 suggests that both interact with phospholipid membranes, changing their conformation. NTPase activity of both proteins was stimulated by the addition of certain phospholipids. According to our results, Virb11-like proteins seem to most likely be involved in the assembly of the membrane-spanning multiprotein complex.

Sequence-related protein export NTPases encoded by the conjugative transfer region of RP4 and by the cag pathogenicity island of Helicobacter pylori share similar hexameric ring structures.

RP4 TrbB, an essential component of the conjugative transfer apparatus of the broad-host-range plasmid RP4, is a member of the PulE protein superfamily involved in multicomponent machineries transporting macromolecules across the bacterial envelope. PulE-like proteins share several well conserved motifs, most notable a nucleoside triphosphate binding motif (P-loop). Helicobacter pylori HP0525 also belongs to the PulE superfamily and is encoded by the pathogenicity island cag, involved in the inflammatory response of infected gastric epithelial cells in mammals. The native molecular masses of TrbB and HP0525 as determined by gel filtration and glycerol gradient centrifugation suggested a homohexameric structure in the presence of ATP and Mg(2+). In the absence of nucleotides and bivalent cations, TrbB behaved as a tetramer whereas the hexameric state of HP0525 remained unaffected. Electron microscopy and image processing demonstrated that TrbB and HP0525 form ring-shaped complexes (diameter: 12 nm) with a central region (diameter: 3 nm) of low electron density when incubated in the presence of ATP and Mg(2+). However, the TrbB average image appeared to be more elliptical with strong twofold rotational symmetry whereas HP0525 complexes are regular hexagons. Six well defined triangle-shaped areas of high electron density were distinguishable in both cases. Covalent crosslinking of TrbB suggests that the hexameric ring is composed from a trimer of dimers, because only dimeric, tetrameric, and hexameric species were detectable. The toroidal structure of TrbB and HP0525 suggests that both proteins catalyze a repetitive process, most probably translocating a cognate substrate across the inner membrane.

TraG from RP4 and TraG and VirD4 from Ti plasmids confer relaxosome specificity to the conjugal transfer system of pTiC58.

Plasmid conjugation systems are composed of two components, the DNA transfer and replication system, or Dtr, and the mating pair formation system, or Mpf. During conjugal transfer an essential factor, called the coupling protein, is thought to interface the Dtr, in the form of the relaxosome, with the Mpf, in the form of the mating bridge. These proteins, such as TraG from the IncP1 plasmid RP4 (TraG(RP4)) and TraG and VirD4 from the conjugal transfer and T-DNA transfer systems of Ti plasmids, are believed to dictate specificity of the interactions that can occur between different Dtr and Mpf components. The Ti plasmids of Agrobacterium tumefaciens do not mobilize vectors containing the oriT of RP4, but these IncP1 plasmid derivatives lack the trans-acting Dtr functions and TraG(RP4). A. tumefaciens donors transferred a chimeric plasmid that contains the oriT and Dtr genes of RP4 and the Mpf genes of pTiC58, indicating that the Ti plasmid mating bridge can interact with the RP4 relaxosome. However, the Ti plasmid did not mobilize transfer from an IncQ relaxosome. The Ti plasmid did mobilize such plasmids if TraG(RP4) was expressed in the donors. Mutations in traG(RP4) with defined effects on the RP4 transfer system exhibited similar phenotypes for Ti plasmid-mediated mobilization of the IncQ vector. When provided with VirD4, the tra system of pTiC58 mobilized plasmids from the IncQ relaxosome. However, neither TraG(RP4) nor VirD4 restored transfer to a traG mutant of the Ti plasmid. VirD4 also failed to complement a traG(RP4) mutant for transfer from the RP4 relaxosome or for RP4-mediated mobilization from the IncQ relaxosome. TraG(RP4)-mediated mobilization of the IncQ plasmid by pTiC58 did not inhibit Ti plasmid transfer, suggesting that the relaxosomes of the two plasmids do not compete for the same mating bridge. We conclude that TraG(RP4) and VirD4 couples the IncQ but not the Ti plasmid relaxosome to the Ti plasmid mating bridge. However, VirD4 cannot couple the IncP1 or the IncQ relaxosome to the RP4 mating bridge. These results support a model in which the coupling proteins specify the interactions between Dtr and Mpf components of mating systems.

Components of the RP4 conjugative transfer apparatus form an envelope structure bridging inner and outer membranes of donor cells: implications for related macromolecule transport systems.

During bacterial conjugation, the single-stranded DNA molecule is transferred through the cell envelopes of the donor and the recipient cell. A membrane-spanning transfer apparatus encoded by conjugative plasmids has been proposed to facilitate protein and DNA transport. For the IncPalpha plasmid RP4, a thorough sequence analysis of the gene products of the transfer regions Tra1 and Tra2 revealed typical features of mainly inner membrane proteins. We localized essential RP4 transfer functions to Escherichia coli cell fractions by immunological detection with specific polyclonal antisera. Each of the gene products of the RP4 mating pair formation (Mpf) system, specified by the Tra2 core region and by traF of the Tra1 region, was found in the outer membrane fraction with one exception, the TrbB protein, which behaved like a soluble protein. The membrane preparation from Mpf-containing cells had an additional membrane fraction whose density was intermediate between those of the cytoplasmic and outer membranes, suggesting the presence of attachment zones between the two E. coli membranes. The Tra1 region is known to encode the components of the RP4 relaxosome. Several gene products of this transfer region, including the relaxase TraI, were detected in the soluble fraction, but also in the inner membrane fraction. This indicates that the nucleoprotein complex is associated with and/or assembled facing the cytoplasmic site of the E. coli cell envelope. The Tra1 protein TraG was predominantly localized to the cytoplasmic membrane, supporting its potential role as an interface between the RP4 Mpf system and the relaxosome.

Helicases as nucleic acid unwinding machines.

Nucleic acid unwinding is an essential step in many biomolecular processes. Researchers in this field recently met to examine progress and outstanding issues.

Crystal structure of the hexameric traffic ATPase of the Helicobacter pylori type IV secretion system.

The type IV secretion system of Helicobacter pylori consists of 10--15 proteins responsible for transport of the transforming protein CagA into target epithelial cells. Secretion of CagA crucially depends on the hexameric ATPase, HP0525, a member of the VirB11-PulE family. We present the crystal structure of a binary complex of HP0525 bound to ADP. Each monomer consists of two domains formed by the N- and C-terminal halves of the sequence. ADP is bound at the interface between the two domains. In the hexamer, the N- and C-terminal domains form two rings, which together form a chamber open on one side and closed on the other. A model is proposed in which HP0525 functions as an inner membrane pore, the closure and opening of which is regulated by ATP binding and ADP release.

Conjugative pili of IncP plasmids, and the Ti plasmid T pilus are composed of cyclic subunits.

TrbC propilin is the precursor of the pilin subunit TrbC of IncP conjugative pili in Escherichia coli. Likewise, its homologue, VirB2 propilin, is processed into T pilin of the Ti plasmid T pilus in Agrobacterium tumefaciens. TrbC and VirB2 propilin are truncated post-translationally at the N terminus by the removal of a 36/47-residue leader peptide, respectively. TrbC propilin undergoes a second processing step by the removal of 27 residues at the C terminus by host-encoded functions followed by the excision of four additional C-terminal residues by a plasmid-borne serine protease. The final product TrbC of 78 residues is cyclized via an intramolecular covalent head-to-tail peptide bond. The T pilin does not undergo additional truncation but is likewise cyclized. The circular structures of these pilins, as verified by mass spectrometry, represent novel primary configurations that conform and assemble into the conjugative apparatus.

Import of DNA into mammalian nuclei by proteins originating from a plant pathogenic bacterium.

Import of DNA into mammalian nuclei is generally inefficient. Therefore, one of the current challenges in human gene therapy is the development of efficient DNA delivery systems. Here we tested whether bacterial proteins could be used to target DNA to mammalian cells. Agrobacterium tumefaciens, a plant pathogen, efficiently transfers DNA as a nucleoprotein complex to plant cells. Agrobacterium-mediated T-DNA transfer to plant cells is the only known example for interkingdom DNA transfer and is widely used for plant transformation. Agrobacterium virulence proteins VirD2 and VirE2 perform important functions in this process. We reconstituted complexes consisting of the bacterial virulence proteins VirD2, VirE2, and single-stranded DNA (ssDNA) in vitro. These complexes were tested for import into HeLa cell nuclei. Import of ssDNA required both VirD2 and VirE2 proteins. A VirD2 mutant lacking its C-terminal nuclear localization signal was deficient in import of the ssDNA-protein complexes into nuclei. Import of VirD2-ssDNA-VirE2 complexes was fast and efficient, and was shown to depended on importin alpha, Ran, and an energy source. We report here that the bacterium-derived and plant-adapted protein-DNA complex, made in vitro, can be efficiently imported into mammalian nuclei following the classical importin-dependent nuclear import pathway. This demonstrates the potential of our approach to enhance gene transfer to animal cells.

Complete sequence of the IncPbeta plasmid R751: implications for evolution and organisation of the IncP backbone.

The broad host range IncP plasmids are of particular interest because of their ability to promote gene spread between diverse bacterial species. To facilitate study of these plasmids we have compiled the complete sequence of the IncPbeta plasmid R751. Comparison with the sequence of the IncPalpha plasmids confirms the conservation of the IncP backbone of replication, conjugative transfer and stable inheritance functions between the two branches of this family. As in the IncPalpha genome the DNA of this backbone appears to have been enriched for the GCCG/CGGC motifs characteristic of the genome of organisms with a high G+C content, such as P. aeruginosa, suggesting that IncPbeta plasmids have been subjected during their evolution to similar mutational and selective forces as IncPalpha plasmids and may have evolved in pseudomonad hosts. The IncP genome is consistently interrupted by insertion of phenotypic markers and/or transposable elements between oriV and trfA and between the tra and trb operons. The R751 genome reveals a family of repeated sequences in these regions which may form the basis of a hot spot for insertion of foreign DNA. Sequence analysis of the cryptic transposon Tn4321 revealed that it is not a member of the Tn21 family as we had proposed previously from an inspection of its ends. Rather it is a composite transposon defined by inverted repeats of a 1347 bp IS element belonging to a recently discovered family which is distributed throughout the prokaryotes. The central unique region of Tn4321 encodes two predicted proteins, one of which is a regulatory protein while the other is presumably responsible for an as yet unidentified phenotype. The most striking feature of the IncPalpha plasmids, the global regulation of replication and transfer by the KorA and KorB proteins encoded in the central control operon, is conserved between the two plasmids although there appear to be significant differences in the specificity of repressor-operator interactions. The importance of these global regulatory circuits is emphasised by the observation that the operator sequences for KorB are highly conserved even in contexts where the surrounding region, either a protein coding or intergenic sequence, has diverged considerably. There appears to be no equivalent of the parABCDE region which in the IncPalpha plasmids provides multimer resolution, lethality to plasmid-free segregants and active partitioning functions. However, we found that the continuous sector from co-ordinate 0 to 9100 bp, encoding the co-regulated klc and kle operons as well as the central control region, could confer a high degree of segregational stability on a low copy number test vector. Thus R751 appears to exhibit very clearly what was first revealed by study of the IncPalpha plasmids, namely a fully functional co-ordinately regulated set of replication, transfer and stable inheritance functions.

The RepA protein of plasmid RSF1010 is a replicative DNA helicase.

The RepA protein of the mobilizable broad host range plasmid RSF1010 has a key function in its replication. RepA is one of the smallest known helicases. The protein forms a homohexamer of 29,896-Da subunits. A variety of methods were used to analyze the quaternary structure of RepA. Gel filtration and cross-linking experiments demonstrated the hexameric structure, which was confirmed by electron microscopy and image reconstruction. These results agree with recent data obtained from RepA crystals diffracting at 3.5-A resolution (Röleke, D., Hoier, H., Bartsch, C., Umbach, P., Scherzinger, E., Lurz, R., and Saenger, W. (1997) Acta Crystallogr. Sec. D 53, 213-216). The RepA helicase has 5' --> 3' polarity. As do most true replicative helicases, RepA prefers a tailed substrate with an unpaired 3'-tail mimicking a replication fork. Optimal unwinding activity was achieved at the remarkably low pH of 5.5. In the presence of Mg2+ (Mn2+) ions, the RepA activity is fueled by ATP, dATP, GTP, and dGTP and less efficiently by CTP and dCTP. UTP and dTTP are poor effectors. Nonhydrolyzable ATP analogues, ADP, and pyrophosphate inhibit the helicase activity, whereas inorganic phosphate does not. The presence of Escherichia coli single-stranded DNA-binding protein stimulates unwinding at physiological pH 2-3-fold, whereas the RSF1010 replicon-specific primase, RepB' protein, has no effect, either in the presence or in the absence of single-stranded DNA-binding protein.

A specific protease encoded by the conjugative DNA transfer systems of IncP and Ti plasmids is essential for pilus synthesis.

TraF, an essential component of the conjugative transfer apparatus of the broad-host-range plasmid RP4 (IncP), which is located at the periplasmic side of the cytoplasmic membrane, encodes a specific protease. The traF gene products of IncP and Ti plasmids show extensive similarities to prokaryotic and eukaryotic signal peptidases. Mutational analysis of RP4 TraF revealed that the mechanism of the proteolytic cleavage reaction resembles that of signal and LexA-like peptidases. Among the RP4 transfer functions, the product of the Tra2 gene, trbC, was identified as a target for the TraF protease activity. TrbC is homologous to VirB2 of Ti plasmids and thought to encode the RP4 prepilin. The maturation of TrbC involves three processing reactions: (i) the removal of the N-terminal signal peptide by Escherichia coli signal peptidase I (Lep), (ii) a proteolytic cleavage at the C terminus by an as yet unidentified host cell enzyme, and (iii) C-terminal processing by TraF. The third reaction of the maturation process is critical for conjugative transfer, pilus synthesis, and the propagation of the donor-specific bacteriophage PRD1. Thus, cleavage of TrbC by TraF appears to be one of the initial steps in a cascade of processes involved in export of the RP4 pilus subunit and pilus assembly mediated by the RP4 mating pair formation function.

Assembly of a functional phage PRD1 receptor depends on 11 genes of the IncP plasmid mating pair formation complex.

PRD1, a lipid-containing double-stranded DNA bacteriophage, uses the mating pair formation (Mpf) complex encoded by conjugative IncP plasmids as a receptor. Functions responsible for conjugative transfer of IncP plasmids are encoded by two distinct regions, Tra1 and Tra2. Ten Tra2 region gene products (TrbB to TrbL) and one from the Tra1 region (TraF) form the Mpf complex. We carried out a mutational analysis of the PRD1 receptor complex proteins by isolating spontaneous PRD1-resistant mutants. The mutations were distributed among the trb genes in the Tra2 region and accumulated predominantly in three genes, trbC, trbE, and trbL. Three of 307 phage-resistant mutants were weakly transfer proficient. Mutations causing a phage adsorption-deficient, transfer-positive phenotype were analyzed by sequencing.

The IncP plasmid-encoded cell envelope-associated DNA transfer complex increases cell permeability.

IncP-type plasmids are broad-host-range conjugative plasmids. DNA translocation requires DNA transfer-replication functions and additional factors required for mating pair formation (Mpf). The Mpf system is located in the cell membranes and is responsible for DNA transport from the donor to the recipient. The Mpf complex acts as a receptor for IncP-specific phages such as PRD1. In this investigation, we quantify the Mpf complexes on the cell surface by a phage receptor saturation technique. Electrochemical measurements are used to show that the Mpf complex increases cell envelope permeability to lipophilic compounds and ATP. In addition it reduces the ability of the cells to accumulate K+. However, the Mpf complex does not dissipate the membrane voltage. The Mpf complex is rapidly disassembled when intracellular ATP concentration is decreased, as measured by a PRD1 adsorption assay.

The helicase domain of phage P4 alpha protein overlaps the specific DNA binding domain.

Replication initiation depends on origin recognition, helicase, and primase activities. In phage P4, a second DNA region, the cis replication region (crr), is also required for replication initiation. The multifunctional alpha protein of phage P4, which is essential for DNA replication, combines the three aforementioned activities on a single polypeptide chain. Protein domains responsible for the activities were identified by mutagenesis. We show that mutations of residues G506 and K507 are defective in vivo in phage propagation and in unwinding of a forked helicase substrate. This finding indicates that the proposed P loop is essential for helicase activity. Truncations of gene product alpha (gp alpha) demonstrated that 142 residues of the C terminus are sufficient for specifically binding ori and crr DNA. The minimal binding domain retains gp alpha's ability to induce loop formation between ori and crr. In vitro and in vivo analysis of short C-terminal truncations indicate that the C terminus is needed for helicase activity as well as for specific DNA binding.

Cnr protein, the negative regulator of bacteriophage P4 replication, stimulates specific DNA binding of its initiator protein alpha.

Bacteriophage P4 DNA replication depends upon the phage-encoded alpha protein, which has DNA helicase and DNA primase activity and can specifically bind to the replication origin (ori) and to the cis replicating region (crr). The P4 Cnr protein functions as a negative regulator of P4 replication, and P4 does not replicate in cells that overexpress cnr. We searched for P4 mutants that suppressed this phenotype (Cnr resistant [alpha cr]). Eight independent mutants that grew in the presence of high levels of Cnr were obtained. None of these can establish the plasmid state. Each of these mutations lies in the DNA binding domain of gp alpha that occupies the C terminus of the protein. Five different sequence changes were found: T675M, G732V (three times), G732W (twice), L733V, and L737V. A TrxA-Cnr fusion protein does not bind DNA by itself but stimulates the ori and crr binding abilities of alpha protein in vitro. The alpha cr mutant proteins were still able to bind specifically to ori or crr, but specific DNA binding was less stimulated by the TrxA-Cnr protein. We present evidence that Cnr protein interacts with the gp alpha domain that binds specifically to DNA and that gp(alpha)cr mutations impair this interaction. We hypothesize that gp alpha-Cnr interaction is essential for the control of P4 DNA replication.