Helix Bundle Domain of Primase RepB' Is Required for Dinucleotide Formation and Extension.

During DNA replication, primases synthesize oligonucleotide primers on single-stranded template DNA, which are then extended by DNA polymerases to synthesize a complementary DNA strand. Primase RepB' of plasmid RSF1010 initiates DNA replication on two 40 nucleotide-long inverted repeats, termed ssiA and ssiB, within the oriV of RSF1010. RepB' consists of a catalytic domain and a helix bundle domain, which are connected by long α-helix 6 and an unstructured linker. Previous work has demonstrated that RepB' requires both domains for the initiation of dsDNA synthesis in DNA replication assays. However, the precise functions of these two domains in primer synthesis have been unknown. Here, we report that both domains of RepB' are required to synthesize a 10-12 nucleotide-long DNA primer, whereas the isolated domains are inactive. Mutational analysis of the catalytic domain indicates that the solvent-exposed W50 plays a critical role in resolving hairpin structures formed by ssiA and ssiB. Three structurally conserved aspartates (D77, D78, and D134) of RepB' catalyze the nucleotidyl transfer reaction. Mutations on the helix bundle domain are identified that either reduce the primer length to a dinucleotide (R285A) or abolish the primer synthesis (D238A), indicating that the helix bundle domain is required to form and extend the initial dinucleotide synthesized by the catalytic domain.

Binding Specificities of the Telomere Phage ϕKO2 Prophage Repressor CB and Lytic Repressor Cro.

Temperate bacteriophages possess a genetic switch which regulates the lytic and lysogenic cycle. The genomes of the temperate telomere phages N15, PY54, and ϕKO2 harbor a primary immunity region (immB) comprising genes for the prophage repressor (cI or cB), the lytic repressor (cro) and a putative antiterminator (q). The roles of these products are thought to be similar to those of the lambda proteins CI (CI prophage repressor), Cro (Cro repressor), and Q (antiterminator Q), respectively. Moreover, the gene order and the location of several operator sites in the prototype telomere phage N15 and in ϕKO2 are reminiscent of lambda-like phages. We determined binding sites of the ϕKO2 prophage repressor CB and lytic repressor Cro on the ϕKO2 genome in detail by electrophoretic mobility shift assay (EMSA) studies. Unexpectedly, ϕKO2 CB and Cro revealed different binding specificities. CB was bound to three OR operators in the intergenic region between cB and cro, two OL operators between cB and the replication gene repA and even to operators of N15. Cro bound exclusively to the 16 bp operator site OR3 upstream of the ϕKO2 prophage repressor gene. The ϕKO2 genes cB and cro are regulated by several strong promoters overlapping with the OR operators. The data suggest that Cro represses cB transcription but not its own synthesis, as already reported for PY54 Cro. Thus, not only PY54, but also phage ϕKO2 possesses a genetic switch that diverges significantly from the switch of lambda-like phages.

The Molecular Switch of Telomere Phages: High Binding Specificity of the PY54 Cro Lytic Repressor to a Single Operator Site.

Temperate bacteriophages possess a molecular switch, which regulates the lytic and lysogenic growth. The genomes of the temperate telomere phages N15, PY54 and ɸKO2 harbor a primary immunity region (immB) comprising genes for the prophage repressor, the lytic repressor and a putative antiterminator. The roles of these products are thought to be similar to those of the lambda proteins CI, Cro and Q, respectively. Moreover, the gene order and the location of several operator sites in the prototype telomere phage N15 and in ɸKO2 are also reminiscent of lambda-like phages. By contrast, in silico analyses revealed the presence of only one operator (O\(_{\rm{R}}\)3) in PY54. The purified PY54 Cro protein was used for EMSA studies demonstrating that it exclusively binds to a 16-bp palindromic site (O\(_{\rm{R}}\)3) upstream of the prophage repressor gene. The O\(_{\rm{R}}\)3 operator sequences of PY54 and ɸKO2/N15 only differ by their peripheral base pairs, which are responsible for Cro specificity. PY54 cI and cro transcription is regulated by highly active promoters initiating the synthesis of a homogenious species of leaderless mRNA. The location of the PY54 Cro binding site and of the identified promoters suggests that the lytic repressor suppresses cI transcription but not its own synthesis. The results indicate an unexpected diversity of the growth regulation mechanisms in lambda-related phages.

The pYV virulence plasmids of Yersinia pseudotuberculosis and Y. pestis contain a conserved DNA region responsible for the mobilization by the self-transmissible plasmid pYE854.

Pathogenic Yersinia strains possess a 70 kb virulence plasmid which can be transmitted to other strains by conjugation systems expressed by co-resident plasmids. We isolated a 720 bp fragment of the Yersinia pseudotuberculosis virulence plasmid p1340 that mediated mobilization of the vector pIV2 by the self-transmissible plasmid pYE854. The p1340 mobilization region contains the resolvase gene tnpR and its proposed resolution site res. Both elements are required for mobilization. Plasmid transfer was associated with the formation of co-integrates in which res was fused to pYE854 fragments by short nucleotide stretches similarly present within res. blast searches and PCR experiments revealed the presence of the mobilization region in the virulence plasmids of other Y. pseudotuberculosis and Y. pestis strains but not in pYV of Yersinia enterocolitica.

Entry exclusion and oriT of a conjugative system encoded by the cryptic plasmid p29930 of Yersinia enterocolitica.

The conjugative transfer system of Yersinia enterocolitica 29930 present on the cryptic plasmid p29930 comprises a mating pore formation system (Mpf) related to that of the IncX plasmid R6K and a DNA transfer and replication system (Dtr) with close relationship to the mob region of the mobilizable plasmid CloDF13. Two regions of the transfer system were selected for more detailed analyses of basic functions of conjugative transfer. The putative open reading frame orf22 located in the Mpf region confers the entry exclusion phenotype to possible recipient cells and inhibited conjugative transfer, when it was inserted into the coding region of the cat gene of pACYC184 in sense direction. Mobilization experiments with recombinant plasmids revealed that a 611bp fragment of the Dtr region containing two repeat sequences were required for a functional oriT by the conjugation system of Y. enterocolitica. While the conjugative transfer of cryptic plasmids harbouring the complete conjugation system had not been demonstrated previously, plasmid pBK17 containing the functional oriT was successfully mobilized from Y. enterocolitica strains into Escherichia coli, thus proving that the transfer system could contribute to the spread of these plasmids in nature.

Structure and function of primase RepB' encoded by broad-host-range plasmid RSF1010 that replicates exclusively in leading-strand mode.

For the initiation of DNA replication, dsDNA is unwound by helicases. Primases then recognize specific sequences on the template DNA strands and synthesize complementary oligonucleotide primers that are elongated by DNA polymerases in leading- and lagging-strand mode. The bacterial plasmid RSF1010 provides a model for the initiation of DNA replication, because it encodes the smallest known primase RepB' (35.9 kDa), features only 1 single-stranded primase initiation site on each strand (ssiA and ssiB, each 40 nt long with 5'- and 3'-terminal 6 and 13 single-stranded nucleotides, respectively, and nucleotides 7-27 forming a hairpin), and is replicated exclusively in leading strand mode. We present the crystal structure of full-length dumbbell-shaped RepB' consisting of an N-terminal catalytic domain separated by a long alpha-helix and tether from the C-terminal helix-bundle domain and the structure of the catalytic domain in a specific complex with the 6 5'-terminal single-stranded nucleotides and the C7-G27 base pair of ssiA, its single-stranded 3'-terminus being deleted. The catalytic domains of RepB' and the archaeal/eukaryotic family of Pri-type primases share a common fold with conserved catalytic amino acids, but RepB' lacks the zinc-binding motif typical of the Pri-type primases. According to complementation studies the catalytic domain shows primase activity only in the presence of the helix-bundle domain. Primases that are highly homologous to RepB' are encoded by broad-host-range IncQ and IncQ-like plasmids that share primase initiation sites ssiA and ssiB and high sequence identity with RSF1010.

Crystal structure of KorA bound to operator DNA: insight into repressor cooperation in RP4 gene regulation.

KorA is a global repressor in RP4 which regulates cooperatively the expression of plasmid genes whose products are involved in replication, conjugative transfer and stable inheritance. The structure of KorA bound to an 18-bp DNA duplex that contains the symmetric operator sequence and incorporates 5-bromo-deoxyuridine nucleosides has been determined by multiple-wavelength anomalous diffraction phasing at 1.96-A resolution. KorA is present as a symmetric dimer and contacts DNA via a helix-turn-helix motif. Each half-site of the symmetric operator DNA binds one copy of the protein in the major groove. As confirmed by mutagenesis, recognition specificity is based on two KorA side chains forming hydrogen bonds to four bases within each operator half-site. KorA has a unique dimerization module shared by the RP4 proteins TrbA and KlcB. We propose that these proteins cooperate with the global RP4 repressor KorB in a similar manner via this dimerization module and thus regulate RP4 inheritance.

Genetic and functional properties of the self-transmissible Yersinia enterocolitica plasmid pYE854, which mobilizes the virulence plasmid pYV.

Yersinia strains frequently harbor plasmids, of which the virulence plasmid pYV, indigenous in pathogenic strains, has been thoroughly characterized during the last decades. Yet, it has been unknown whether the nonconjugative pYV can be transferred by helper plasmids naturally occurring in this genus. We have isolated the conjugative plasmids pYE854 (95.5 kb) and pYE966 (70 kb) from a nonpathogenic and a pathogenic Yersinia enterocolitica strain, respectively, and demonstrate that both plasmids are able to mobilize pYV. The complete sequence of pYE854 has been determined. The transfer proteins and oriT of the plasmid reveal similarities to the F factor. However, the pYE854 replicon does not belong to the IncF group and is more closely related to a plasmid of gram-positive bacteria. Plasmid pYE966 is very similar to pYE854 but lacks two DNA regions of the larger plasmid that are dispensable for conjugation.

Unsaturated fatty acids are inhibitors of bacterial conjugation.

This report describes a high-throughput assay to identify substances that reduce the frequency of conjugation in Gram-negative bacteria. Bacterial conjugation is largely responsible for the spread of multiple antibiotic resistances in human pathogens. Conjugation inhibitors may provide a means to control the spread of antibiotic resistance. An automated conjugation assay was developed that used plasmid R388 and a laboratory strain of Escherichia coli as a model system, and bioluminescence as a reporter for conjugation activity. Frequencies of conjugation could be measured continuously in real time by the amount of light produced, and thus the effects of inhibitory compounds could be determined quantitatively. A control assay, run in parallel, allowed elimination of compounds affecting cell growth, plasmid stability or gene expression. The automated conjugation assay was used to screen a database of more than 12,000 microbial extracts known to contain a wide variety of bioactive compounds (the NatChem library). The initial hit rate was 1.4 %. From these, 48 extracts containing active compounds and representing a variety of organisms and extraction conditions were subjected to fractionation (24 fractions per extract). The 52 most active fractions were subjected to a secondary analysis to determine the range of plasmid inhibition. Plasmids R388, R1 and RP4 were used as representatives of a variety of plasmid transfer systems. Only one fraction (of complex composition) affected transfer of all three plasmids, while four other fractions were active against two of them. Two separate compounds were identified from these fractions: linoleic acid and dehydrocrepenynic acid. Downstream analysis showed that the chemical class of unsaturated fatty acids act as true inhibitors of conjugation.

Introduction of DNA into Actinomycetes by bacterial conjugation from E. coli--an evaluation of various transfer systems.

Gene transfer is a basic requirement for optimizing bioactive natural substances produced by an increasing number of industrially used microorganisms. We have analyzed quantitatively horizontal gene transfer from Escherichia coli to Actinomycetes. The efficiencies of DNA transfer of four different systems were compared that consist of conjugative and mobilizable plasmids with a broad-host range. Three novel binary vector set-ups were constructed based on: (i) the IncQ group of mobilizable plasmids (RSF1010), (ii) IncQ-like pTF-FC2 and (iii) pSB102 that belongs to a new class of broad-host-range plasmids. The established system based on the IncPalpha group of conjugative plasmids served as the reference. For all plasmids constructed, we confirmed the functional integrity of the selected transfer machineries by intrageneric matings between E. coli strains. We demonstrate that the transfer systems introduced in this study are efficient in mediating gene transfer from E. coli to Actinomycetes and are possible alternatives for gene transfer into Actinomycetes for which the IncPalpha-based transfer system is not applicable. The use of plasmids that integrate into the recipients' chromosomes compared to that of plasmids replicating autonomously is shown to allow the access to a wider range of hosts.

The repA gene of the linear Yersinia enterocolitica prophage PY54 functions as a circular minimal replicon in Escherichia coli.

The Yersinia enterocolitica prophage PY54 replicates as a linear DNA molecule with covalently closed ends. For replication of a circular PY54 minimal replicon that has been derived from a linear minireplicon, two phage-encoded loci are essential in Escherichia coli: (i) the reading frame of the replication initiation gene repA and (ii) its 212-bp origin located within the 3' portion of repA. The RepA protein acts in trans on the origin since we have physically separated the PY54 origin and repA onto a two-plasmid origin test system. For this trans action, the repA 3' end carrying the origin is dispensable. Mutagenesis by alanine scan demonstrated that the motifs for primase and for nucleotide binding present in the protein are essential for RepA activity. The replication initiation functions of RepA are replicon specific. The replication initiation proteins DnaA, DnaG, and DnaB of the host are unable to promote origin replication in the presence of mutant RepA proteins that carry single residue exchanges in these motifs. The proposed origins of the known related hairpin prophages PY54, N15, and PKO2 are all located toward the 3' end of the corresponding repA genes, where several structure elements are conserved. Origin function depends on the integrity of these elements.

The mating pair formation system of conjugative plasmids-A versatile secretion machinery for transfer of proteins and DNA.

The mating pair formation (Mpf) system functions as a secretion machinery for intercellular DNA transfer during bacterial conjugation. The components of the Mpf system, comprising a minimal set of 10 conserved proteins, form a membrane-spanning protein complex and a surface-exposed sex pilus, which both serve to establish intimate physical contacts with a recipient bacterium. To function as a DNA secretion apparatus the Mpf complex additionally requires the coupling protein (CP). The CP interacts with the DNA substrate and couples it to the secretion pore formed by the Mpf system. Mpf/CP conjugation systems belong to the family of type IV secretion systems (T4SS), which also includes DNA-uptake and -release systems, as well as effector protein translocation systems of bacterial pathogens such as Agrobacterium tumefaciens (VirB/VirD4) and Helicobacter pylori (Cag). The increased efforts to unravel the molecular mechanisms of type IV secretion have largely advanced our current understanding of the Mpf/CP system of bacterial conjugation systems. It has become apparent that proteins coupled to DNA rather than DNA itself are the actively transported substrates during bacterial conjugation. We here present a unified and updated view of the functioning and the molecular architecture of the Mpf/CP machinery.

Protein circlets as sex pilus subunits.

The largest circular protein structures discovered define a class of transfer proteins acting in bacterial conjugation and type IV secretion. Proteins ranging from 73 to 78 residues with head-to-tail peptide bonds constitute the major subunit of conjugative pili of some type IV secretion systems. Their plasmid-encoded precursors are enzymatically processed and cyclized before being assembled into pili. These extra-cellular surface filaments mediate physical contact between donor and recipient cell or pathogen and host cell. Pili are essential prerequisites for DNA and protein transfer. A membrane-bound signal peptidase-like enzyme is responsible for the circularization reaction. Site-directed mutagenesis and mass spectrometry has been used extensively to unravel the mechanism of the enzyme-substrate interaction of the pilin maturation process.

Sequence-specific DNA binding determined by contacts outside the helix-turn-helix motif of the ParB homolog KorB.

The KorB protein of the broad-host-range plasmid RP4 acts as a multifunctional regulator of plasmid housekeeping genes, including those responsible for replication, maintenance and conjugation. Additionally, KorB functions as the ParB analog of the plasmid's partitioning system. The protein structure consists of eight helices, two of which belong to a predicted helix-turn-helix motif. Each half-site of the palindromic operator DNA binds one copy of the protein in the major groove. As confirmed by mutagenesis, recognition specificity is based mainly on two side chain interactions outside the helix-turn-helix motif with two bases next to the central base pair of the 13-base pair operator sequence. The surface of the KorB DNA-binding domain mirrors the overall acidity of KorB, whereas DNA binding occurs via a basic interaction surface. We present a model of KorB, including the structure of its dimerization domain, and discuss its interactions with the highly basic ParA homolog IncC.

RP4 repressor protein KorB binds to the major groove of the operator DNA: a Raman study.

KorB is a member of the ParB family of bacterial partitioning proteins. The protein encoded by the conjugative plasmid RP4 is part of the global control circuit and regulates the expression of plasmid genes, the products of which are involved in replication, transfer, and stable inheritance. KorB is a homodimeric protein which binds to palindromic 13 bp DNA sequences [5'-TTTAGC((G)/(C))GCTAAA-3'] present 12 times in the 60 kb plasmid. Each KorB subunit is composed of two domains; the C-domain is responsible for the dimerization of the protein, whereas the N-terminal domain recognizes and binds to the operator sequence (O(B)). Here we describe results of a Raman spectroscopic study of the interaction of the N-domain with a double-stranded model oligonucleotide composed of the palindromic binding sequence and terminal 5'-A(Br)U and AG-3' bases. Comparison of the Raman spectra of the free KorB N-domain and O(B) DNA with the spectrum of the complex reveals large differences. KorB-N binds in the major groove of the O(B) DNA, and the interactions induce changes in the DNA backbone and in the secondary structure of the protein.

Hexameric RSF1010 helicase RepA: the structural and functional importance of single amino acid residues.

In the known monoclinic crystals the 3-dimensional structure of the hexameric, replicative helicase RepA encoded by plasmid RSF1010 shows 6-fold rotational symmetry. In contrast, in the cubic crystal form at 2.55 A resolution described here RepA has 3-fold symmetry and consists of a trimer of dimers. To study structure-function relationships, a series of repA deletion mutants and mutations yielding single amino acid exchanges were constructed and the respective gene products were analyzed in vivo and in vitro. Hexamerization of RepA occurs via the N-terminus and is required for NTP hydrolysis. The C-terminus is essential both for the interaction with the replication machinery and for the helicase activity. Functional analyses of RepA variants with single amino acid exchanges confirmed most of the predictions that were based on the published 3-dimensional structure. Of the five motifs conserved in family 4 helicases, all residues conserved in RepA and T7 gp4 helicases participate in DNA unwinding. Residues K42, E76, D77, D139 and H178, proposed to play key roles in catalyzing the hydrolysis of NTPs, are essential for RepA activity. Residue H178 of motif H3 couples nucleotide consumption to DNA strand separation.

A cryptic plasmid of Yersinia enterocolitica encodes a conjugative transfer system related to the regions of CloDF13 Mob and IncX Pil.

Yersinia enterocolitica 29930 (biotype 1A; O : 7,8), the producing strain of the phage-tail-like bacteriocin enterocoliticin, possesses a plasmid-encoded conjugative type IV transfer system. The genes of the conjugative system were found by screening of a cosmid library constructed from total DNA of strain 29930. The cosmid Cos100 consists of the vector SuperCos1 and an insert DNA of 40 303 bp derived from a cryptic plasmid of strain 29930. The conjugative transfer system consists of genes encoding a DNA transfer and replication system (Dtr) with close relationship to the mob region of the mobilizable plasmid CloDF13 and a gene cluster encoding a mating pair formation system (Mpf) closely related to the Mpf system of the IncX plasmid R6K. However, a gene encoding a homologue of TaxB, the coupling protein of the IncX system, is missing. The whole transfer region has a size of approximately 17 kb. The recombinant plasmid Cos100 was shown to be transferable between Escherichia coli and Yersinia with transfer frequencies up to 0.1 transconjugants per donor. Mutations generated by inserting a tetracycline cassette into putative tri genes yielded a transfer-deficient phenotype. Conjugative transfer of the cryptic plasmid could not be demonstrated in the original host Y. enterocolitica 29930. However, a kanamycin-resistance-conferring derivative of the plasmid was successfully introduced into E. coli K-12 by transformation and was shown to be self-transmissible. Furthermore, Southern blot hybridization and PCR experiments were carried out to elucidate the distribution of the conjugative transfer system in Yersinia. In total, six Y. enterocolitica biotype 1A strains harbouring closely related systems on endogenous plasmids were identified.

Bacteriophage P1 Ban protein is a hexameric DNA helicase that interacts with and substitutes for Escherichia coli DnaB.

Since the ban gene of bacteriophage P1 suppresses a number of conditionally lethal dnaB mutations in Escherichia coli, it was assumed that Ban protein is a DNA helicase (DnaB analogue) that can substitute for DnaB in the host replication machinery. We isolated and sequenced the ban gene, purified the product, and analysed the function of Ban protein in vitro and in vivo. Ban hydrolyses ATP, unwinds DNA and forms hexamers in the presence of ATP and magnesium ions. Since all existing conditionally lethal dnaB strains bear DnaB proteins that may interfere with the protein under study, we constructed a dnaB null strain by using a genetic set-up designed to provoke the conditional loss of the entire dnaB gene from E.coli cells. This novel tool was used to show that Ban restores the viability of cells that completely lack DnaB at 30 degrees C, but not at 42 degrees C. Surprisingly, growth was restored by the dnaB252 mutation at a temperature that is restrictive for ban and dnaB252 taken separately. This indicates that Ban and DnaB are able to interact in vivo. Complementary to these results, we demonstrate the formation of DnaB-Ban hetero-oligomers in vitro by ion exchange chromatography. We discuss the interaction of bacterial proteins and their phage-encoded analogues to fulfil functions that are essential to phage and host growth.

TraG-like proteins of type IV secretion systems: functional dissection of the multiple activities of TraG (RP4) and TrwB (R388).

TraG-like proteins are essential components of type IV secretion systems. During secretion, TraG is thought to translocate defined substrates through the inner cell membrane. The energy for this transport is presumably delivered by its potential nucleotide hydrolase (NTPase) activity. TraG of conjugative plasmid RP4 is a membrane-anchored oligomer that binds RP4 relaxase and DNA. TrwB (R388) is a hexameric TraG-like protein that binds ATP. Both proteins, however, lack NTPase activity under in vitro conditions. We characterized derivatives of TraG and TrwB truncated by the N-terminal membrane anchor (TraGdelta2 and TrwBdelta1) and/or containing a point mutation at the putative nucleotide-binding site (TraGdelta2K187T and TraGK187T). Unlike TraG and TrwB, truncated derivatives behaved as monomers without the tendency to form oligomers or aggregates. Surface plasmon resonance analysis with immobilized relaxase showed that mutant TraGK187T was as good a binding partner as the wild-type protein, whereas truncated TraG monomers were unable to bind relaxase. TraGdelta2 and TrwBdelta1 bound ATP and, with similar affinity, ADP. Binding of ATP and ADP was strongly inhibited by the presence of Mg(2+) or single-stranded DNA and was competed for by other nucleotides. Compared to the activity of TraGdelta2, the ATP- and ADP-binding activity of the point mutation derivative TraGdelta2K187T was significantly reduced. Each TraG derivative bound DNA with an affinity similar to that of the native protein. DNA binding was inhibited or competed for by ATP, ADP, and, most prominently, Mg(2+). Thus, both nucleotide binding and DNA binding were sensitive to Mg(2+) and were competitive with respect to each other.

PY54, a linear plasmid prophage of Yersinia enterocolitica with covalently closed ends.

PY54 is a temperate phage isolated from Yersinia enterocolitica. Lysogenic Yersinia strains harbour the PY54 prophage as a plasmid (pY54). The plasmid has the same size (46 kb) as the PY54 genome isolated from phage particles. By electron microscopy, restriction analysis and DNA sequencing, it was demonstrated that the phage and the plasmid DNAs are linear, circularly permuted molecules. Unusually for phages of Gram-negative bacteria, the phage genome has 3'-protruding ends. The linear plasmid pY54 has covalently closed ends forming telomere-like hairpins. The equivalent DNA sequence of the phage genome is a 42 bp perfect palindrome. Downstream from the palindrome, an open reading frame (ORF) was identified that revealed strong DNA homology to the telN gene of Escherichia coli phage N15 encoding a protelomerase. Similar to PY54, the N15 prophage is a linear plasmid with telomeres. The N15 protelomerase has cleaving/joining activity generating the telomeres by processing a 56 bp palindrome (telomere resolution site tel RL). To study the activity of the PY54 protein, the telN-like gene was cloned and expressed in E. coli. A 77 kDa protein was obtained and partially purified. The protein was found to process recombinant plasmids containing the 42 bp palindrome. Telomere resolution of plasmids under in vivo conditions was also investigated in Yersinia infected with PY54. Processing required a plasmid containing the palindrome as well as adjacent DNA sequences from the phage including an additional inverted repeat. Regions on the phage genome important for plasmid maintenance were defined by the construction of linear and circular miniplasmid derivatives of pY54, of which the smallest miniplasmid comprises a 4.5 kb DNA fragment of the plasmid prophage.