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.

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.

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.

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.

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. Acting on a telomere resolution site telRL, the protelomerase converts circular plasmid DNA into linear covalently closed dumbbell-shaped molecules ("doggybones") in a single-step enzyme reaction. Two such sites were inserted into an expression plasmid flanking a gene of interest. This is cleaved and joined by means of the protelomerase, yielding linear closed mini DNA coding for green fluorescent protein (EGFP) or interleukin-12 (IL-12). Upon transient transfection of human embryonal kidney cells, EGFP was expressed at higher levels from linear closed molecules than from linear open molecules generated by restriction endonucleases for comparison. The level of transcription was comparable to that observed for the parental plasmid DNA. To test whether the linear closed mini DNA molecules are functional in vivo the B16F10/C57BL/6 melanoma metastasis model was applied, where injection of IL-12-expressing DNA inhibits metastasis formation in the lung. The anti-metastatic effect of the IL-12-expressing linear closed DNA was equal or higher than that of the parental plasmid DNA. Therefore, the TelN/ telRL system is well suited to generate linear closed mini DNA with high stability and a minimum of foreign nucleotide sequences.

Phage P4 origin-binding domain structure reveals a mechanism for regulation of DNA-binding activity by homo- and heterodimerization of winged helix proteins.

The origin-binding domain of the gpalpha protein of phage P4 (P4-OBD) mediates origin recognition and regulation of gpalpha activity by the protein Cnr. We have determined the crystal structure of P4-OBD at 2.95 A resolution. The structure of P4-OBD is that of a dimer with pseudo twofold symmetry. Each subunit has a winged helix topology with a unique structure among initiator proteins. The only structural homologue of the P4-OBD subunit is the DNA-binding domain of the eukaryotic transcriptional activator Rfx1. Based on this structural alignment, a model for origin recognition by the P4-OBD dimer is suggested. P4-OBD mutations that interfere with Cnr binding locate to the dimer interface, indicating that Cnr acts by disrupting the gpalpha dimer. P4-OBD dimerization is mediated by helices alpha1 and alpha3 in both subunits, a mode of winged helix protein dimerization that is reminiscent of that of the eukaryotic transcription factors E2F and DP. This, in turn, suggests that Cnr is also a winged helix protein, a possibility that is supported by previously unreported sequence homologies between Cnr and Rfx1 and homology modelling. Hence, in a mechanism that appears to be conserved from phage to man, the DNA-binding activity of winged helix proteins can be regulated by other winged helix proteins via the versatile use of the winged helix motif as a homo- or heterodimerization scaffold.

Phage N15 telomere resolution. Target requirements for recognition and processing by the protelomerase.

The Escherichia coli prophage N15 exists as a linear DNA molecule with covalently closed ends. Purified N15 protelomerase TelN is the only protein required to convert circular DNA substrates to the linear form with hairpin termini. Within the center of the telomerase occupancy site tos, the target for TelN is the 56-bp telRL consisting of the central 22-bp palindrome telO and two 14-bp flanking inverted sequence repetitions. DNase I footprinting of TelN-telRL complexes shows a segment of approximately 50 bp protected by TelN. Surface plasmon resonance studies demonstrate that this extended footprint is caused by two TelN molecules bound to telRL. Stable TelN-target DNA complexes are achieved with telRL; however, the additional sequences of tos stabilize the TelN-target complexes. TelO alone is not sufficient for specific stable complex formation. However, processing can occur, i.e. generation of the linear covalently closed DNA. Within the context of telRL, sequences of telO are involved in specific TelN-telRL complex formation, in processing itself, and/or in recognition of the processing site. The sequence of the central (CG)(3) within telO that is part of a 14-bp stretch proposed to have Z-DNA conformation is essential for processing but not for formation of specific TelN-telRL complexes. The concerted action of both TelN molecules at the target site is the basis for telomere resolution. Capturing of reaction intermediates demonstrates that TelN binds covalently to the 3'-phosphoryl of the cleaved strands.

An Src homology 3-like domain is responsible for dimerization of the repressor protein KorB encoded by the promiscuous IncP plasmid RP4.

KorB is a regulatory protein encoded by the conjugative plasmid RP4 and a member of the ParB family of bacterial partitioning proteins. The protein regulates the expression of plasmid genes whose products are involved in replication, transfer, and stable inheritance of RP4 by binding to palindromic 13-bp DNA sequences (5'-TTTAGC(G/C)GCTAAA-3') present 12 times in the 60-kb plasmid. Here we report the crystal structure of KorB-C, the C-terminal domain of KorB comprising residues 297-358. The structure of KorB-C was solved in two crystal forms. Quite unexpectedly, we find that KorB-C shows a fold closely resembling the Src homology 3 (SH3) domain, a fold well known from proteins involved in eukaryotic signal transduction. From the arrangement of molecules in the asymmetric unit, it is concluded that two molecules form a functionally relevant dimer. The detailed analysis of the dimer interface and a chemical cross-linking study suggest that the C-terminal domain is responsible for stabilizing the dimeric form of KorB in solution to facilitate binding to the palindromic operator sequence. The KorB-C crystal structure extends the range of protein-protein interactions known to be promoted by SH3 and SH3-like domains.