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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 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.

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.

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.

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.

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.

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.

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.

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.

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.

VirB11 ATPases are dynamic hexameric assemblies: new insights into bacterial type IV secretion.

The coupling of ATP binding/hydrolysis to macromolecular secretion systems is crucial to the pathogenicity of Gram-negative bacteria. We reported previously the structure of the ADP-bound form of the hexameric traffic VirB11 ATPase of the Helicobacter pylori type IV secretion system (named HP0525), and proposed that it functions as a gating molecule at the inner membrane, cycling through closed and open forms regulated by ATP binding/hydrolysis. Here, we combine crystal structures with analytical ultracentrifugation experiments to show that VirB11 ATPases indeed function as dynamic hexameric assemblies.

The VirB4 family of proposed traffic nucleoside triphosphatases: common motifs in plasmid RP4 TrbE are essential for conjugation and phage adsorption.

Proteins of the VirB4 family are encoded by conjugative plasmids and by type IV secretion systems, which specify macromolecule export machineries related to conjugation systems. The central feature of VirB4 proteins is a nucleotide binding site. In this study, we asked whether members of the VirB4 protein family have similarities in their primary structures and whether these proteins hydrolyze nucleotides.

Linear closed mini DNA generated by the prokaryotic cleaving-joining enzyme TelN is functional in mammalian cells.

For application of DNA in gene medicine plasmid or viral DNA is usually used as a vector for the gene of interest. To generate DNA with a minimum of foreign DNA sequences, we used the prokaryotic telomerase, protelomerase TelN, of bacteriophage N15. This is a novel enzyme with cleaving-joining activity, which is required for the formation of linear prophage DNA with closed ends in lysogenic bacteria.

Tying rings for sex.

The primary component of the sex pilus encoded by IncP (RP4) and Ti plasmids has been identified as a circular pilin protein with a peptide bond between the amino and carboxyl terminus. Here, we review the key experiments that led to this discovery, and the present mechanistic model for pilin-precursor processing and the cyclization reaction. In addition, we discuss the implications for horizontal gene transfer in bacterial conjugation.

The complete nucleotide sequence and environmental distribution of the cryptic, conjugative, broad-host-range plasmid pIPO2 isolated from bacteria of the wheat rhizosphere.

Plasmid pIPO2 is a cryptic, conjugative, broad-host-range plasmid isolated from the wheat rhizosphere. It efficiently self-transfers between alpha, beta and gamma Proteobacteria and has a mobilizing/retromobilizing capacity for IncQ plasmids. The complete nucleotide sequence of pIPO2 is presented on the basis of its mini-Tn5::luxABtet-tagged derivative, pIPO2T.