Functional and mutational analysis of conjugative transfer region 2 (Tra2) from the IncHI1 plasmid R27

The transfer 2 region (Tra2) of the conjugative plasmid drR27 (derepressed R27) was analyzed by PSI-BLAST, insertional mutagenesis, genetic complementation, and an H-pilus assay. Tra2 contains 11 mating-pair formation (Mpf) genes that are essential for conjugative transfer, 9 of which are essential for H-pilus production (trhA, -L, -E, -K, -B, -V, -C, -P, and -W). TrhK has similarity to secretin proteins, suggesting a mechanism by which DNA could traverse the outer membrane of donors. The remaining two Mpf genes, trhU and trhN, play an auxiliary role in H-pilus synthesis and are proposed to be involved in DNA transfer and mating-pair stabilization, respectively. Conjugative transfer abilities were restored for each mutant when complemented with the corresponding transfer gene. In addition to the essential Mpf genes, three genes, trhO, trhZ, and htdA, modulate R27 transfer frequency. Disruption of trhO and trhZ severely reduced the transfer frequencies of drR27, whereas disruption of htdA greatly increased the transfer frequency of wild-type R27 to drR27 levels. A comparison of the essential transfer genes encoded by the Tra2 and Tra1 (T. D. Lawley, M. W. Gilmour, J. E. Gunton, L. J. Standeven, and D. E. Taylor, J. Bacteriol. 184:2173-2183, 2002) of R27 to other transfer systems illustrates that the R27 conjugative transfer system is a chimera composed of IncF-like and IncP-like transfer systems. Furthermore, the Mpf/type IV secretion systems encoded by IncH and IncF transfer systems are distinct from that of the IncP transfer system. The phenotypic and ecological significance of these observations is discussed.     

Characterization of maxi- and mini-derivatives of the IncHI1 plasmid R27

R27, an IncHI1 plasmid of 182 kilobases (kb), which was originally isolated fromSalmonella typhimurium, was found to contain two copies of IS1 in direct orientation. Deletion derivatives constructed in vitro were of two types: a maxi-derivative of 110 kb (pDT1047) with a single complete copy of IS1, and mini-derivatives of 5–6 kb which contained less than a complete copy of IS1. The IncHI1 miniplasmids also contained a portion of the tetracycline resistance determinant from R27 and a replication region related to the RepFIA replicon. Electron microscopic homoduplex studies demonstrated the presence of two other inverted repeat sequences within the miniplasmid that were unrelated to IS1.     

Functional and mutational analysis of conjugative transfer region 1 (Tra1) from the IncHI1 plasmid R27

The conjugative transfer region 1 (Tra1) of the IncHI1 plasmid R27 was subjected to DNA sequence analysis, mutagenesis, genetic complementation, and an H-pilus-specific phage assay. Analysis of the nucleotide sequence indicated that the Tra1 region contains genes coding for mating pair formation (Mpf) and DNA transfer replication (Dtr) and a coupling protein. Insertional disruptions of 9 of the 14 open reading frames (ORFs) in the Tra1 region resulted in a transfer-deficient phenotype. Conjugative transfer was restored for each transfer mutant by genetic complementation. An intergenic region between traH and trhR was cloned and mobilized by R27, indicating the presence of an origin of transfer (oriT). The five ORFs immediately downstream of the oriT region are involved in H-pilus production, as determined by an H-pilus-specific phage assay. Three of these ORFs encode proteins homologous to Mpf proteins from IncF plasmids. Upstream of the oriT region are four ORFs required for plasmid transfer but not H-pilus production. TraI contains sequence motifs that are characteristic of relaxases from the IncP lineage but share no overall homology to known relaxases. TraJ contains both an Arc repressor motif and a leucine zipper motif. A putative coupling protein, TraG, shares a low level of homology to the TraG family of coupling proteins and contains motifs that are important for DNA transfer. This analysis indicates that the Mpf components of R27 share a common lineage with those of the IncF transfer system, whereas the relaxase of R27 is ancestrally related to that of the IncP transfer system.     

Genetic analysis of transfer and incompatibility functions within the IncHI plasmid R27

This study was undertaken to establish a transfer complementation system for IncH plasmids and to locate regions of incompatibility within the HI1 plasmid, R27. Two regions of R27 were found to contribute to incompatibility as determined by incompatibility testing with fragments of R27 cloned in cosmid vectors. One of these regions hybridized with the IncHI1 rep probe (Couturier et al., Microbiol. Rev. 52, 375-395, 1988). Complementation analysis was carried out using transfer-deficient mutants of R27 in combination with pHH1508a. Cosmid vectors, which contained cloned restriction fragments of R27, were able to complement selected R27 Tra- mutants, enabling the transfer-deficient plasmid to transfer at near-normal frequencies. Complementation of R27 Tra- plasmids by pHH1508a at both 26 and 37 degrees C was shown to occur, but was host-dependent in its degree. These results suggest that the transfer mechanisms of IncHI and IncHII plasmids are related.     

Isolation and location on the R27 map of two replicons and an incompatibility determinant specific for IncHI1 plasmids.

Two replicons were isolated independently from different IncHI1 plasmids. One was isolated from R27, and a second was isolated from pIP522. We demonstrate, by DNA-DNA hybridization experiments, that these maintenance regions are different and that they are specific to, and carried by, all IncHI1 plasmids tested. In view of this specificity we decided to designate the replicon isolated from R27 as RepHI1A and the replicon isolated from pIP522 as RepHI1B. These two autoreplicative regions are not related to a third replicon present in all IncHI1 plasmids that bears homology with RepFIA and that expresses the characteristic incompatibility of IncHI1 subgroup plasmids toward F factor (D. Saul, D. Lane, and P. L. Bergquist, Mol. Microbiol. 2:219-225, 1988; D. E. Taylor, R. W. Hedges, and P. L. Bergquist, J. Gen. Microbiol. 131:1523-1530, 1985). These results demonstrate that all IncHI1 plasmids tested contain at least three replicons. An incompatibility (Inc) region that hybridizes specifically to all the IncHI1 plasmids was previously isolated (M. Couturier, F. Bex, P. L. Bergquist, and W. K. Maas, Microbiol. Rev. 52:375-395, 1988). Although this Inc locus is not located in an autoreplicative region of IncHI1 plasmids, we observed that this locus stabilizes a low-copy-number replicon. This Inc locus is probably a component of an active partition locus involved in the maintenance of IncHI1 plasmids. The nucleotide sequence of the Inc region contains direct repeats of 31 bp. In addition, this incompatibility determinant hybridizes specifically with IncHI1 plasmids but expresses incompatibility toward plasmids of both IncHI subgroups (IncHI1 and IncHI2). In this communication, we present the mapping of these maintenance elements on the R27 genome.     

A replication region of the IncHI plasmid, R27, is highly homologous with the RepFIA replicon of F

A region of the IncHI plasmid R27 has been found to share very close nucleotide sequence homology with the RepFIA replicon of F. This region has been located on a 1.6 kb segment of R27 plasmid DNA, and corresponds to ori-2 and the E gene of F. The incC repeat sequence region shows reduced homology, with the F repeats being an imperfect subset of a larger repeated sequence found in R27. The E gene homologue of R27 is able to initiate replication from the F ori-2 sequence and to repress the E gene promoter of F. The results are consistent with the observed incompatibility behaviour of R27, and have a bearing on the specificity of interaction of E protein with its DNA-binding sites.     

Cloning and genetic analysis of tra cistrons of the Tra 2/Tra 3 region of plasmid RP1

Transfer-defective mutants of the 10.4-kb Tra 2/Tra 3 region of RP1 were identified by their ability to be complemented by clones carrying all or part of this region. The respective mutations occurred in six cistrons whose order (traA, B, E, R, P, Q) and location were determined by deletion and insertion mapping. The cistrons occupy a minimum of 5.5 kb with the most distal, traA, spanning the 28.0-kb map position and traR the KpnI site at map position 24.1 kb. Each cistron is expressed independently, as Tn5 or Tn504 insertions in any one cistron do not affect the other five. The phenotypes controlled by each cistron suggest that all contribute to pilus biosynthesis/function while three (traB, R, and P) also contribute to surface exclusion. Given the occurrence of tra cistrons in the "silent" region between Tra 2 and Tra 3 we propose that the epithet "Tra 2" should be used to describe this entire region.     

Arsenical resistance in the IncHI2 plasmids

The IncHI2 plasmid R478, like other arsenic resistance IncH plasmids, provides increased levels of resistance to sodium arsenate (up to 100mM) and sodium arsenite (up to 10mM) to the host cell. An arsenic resistance fragment of R478 was cloned and sequenced revealing four arsenic resistance associated gene homologues, arsR, arsB, arsC, and arsH. Two other open reading frames in the cloned fragment were found to be homologues of sulphate transport associated genes. Both the four gene arsenic resistance operons and the two gene sulphate transport operons have been previously shown to be transposon associated. However, no evidence of transposability was found associated with these operons in R478. Both the R478 associated arsenic and sulphate transport operons were shown to be common to all arsenic resistance IncH plasmids examined by Southern hybridisation and PCR analysis.     

Characterization and Sequence Analysis of the Replicator Region of the Novel Plasmid pALC1 from Paracoccus alcaliphilus

The replicator region of a low-copy-number plasmid, pALC1, of Paracoccus alcaliphilus JCM 7364 was cloned in a form of the minireplicon pALC100 (3.6 kb). The host range of the minireplicon embraces several species of genus Paracoccus, as well as Agrobacterium tumefaciens, Rhizobium leguminosarum, and Rhodobacter sphaeroides (all belonging to alpha-Proteobacteria), but not Escherichia coli. The complete nucleotide sequence of the replicator region (2276 bp) revealed the presence of one complete open reading frame coding for the 28.4-kDa protein (RepA) with similarity to replication proteins of plasmid pSW500 of Erwinia stewartii and pVS1 of Pseudomonas fluorescens. The iteron-like region was identified upstream of the repA gene and consisted of two clusters of repeated sequences (17 bp long) separated by a putative DnaA box. Analysis of the predicted amino acid sequence of two adjacent incomplete ORFs suggests the localization of repA between genes involved in conjugation (traG) and partitioning (parA) within the pALC1 genome.     

Interaction between the IncHI1 plasmid R27 coupling protein and type IV secretion system: TraG associates with the coiled-coil mating pair formation protein TrhB

Assemblies of plasmid-encoded proteins direct the conjugative transfer of plasmid DNA molecules between bacteria. These include the membrane-associated mating pair formation (Mpf) complex necessary for pilus production and the cytoplasmic relaxosome required for DNA processing. The proposed link between these distinct protein complexes is the coupling protein (the TraG family of proteins). Interactions between the coupling protein and relaxosome components have been previously characterized and we document here, for the first time, a direct interaction between the coupling protein and an Mpf protein. Using the adenylate cyclase bacterial two-hybrid (BTH) system, we present in vivo evidence that the IncHI1 plasmid R27-encoded proteins TraG and TrhB interact. This interaction was verified through a co-immunoprecipitation reaction. We have also been able to delineate the interaction domain of TrhB to TraG by showing a positive interaction using the first 220 amino acids of TrhB (452 aa). TrhB has a proline-rich domain from amino acids 135-173 which may serve to facilitate protein interactions and/or periplasmic extension. TrhB self association was detected using far-Western, co-immunoprecipitation, and also BTH analysis, which was used to define the homotypic interaction domain, comprising a predicted coiled-coil region at residues 77-124 of TrhB. These data support a model in which the coupling protein interacts with an Mpf component to target the transferring DNA strand held by the relaxosome to the transmembrane Mpf complex.     

Cloning the Tra1 region of RP1

The Tra1 region of RP1 from a derivative with Tn7 inserted into the kanamycin resistance determinant was cloned, using EcoRI, into the multicopy vector plasmid pBR325. For one orientation of the cloned fragment the resultant chimeric plasmid was very frequently lost from the cell, but in the other orientation it was much more stable and also compatible with RP1. Complementation by the stable chimeric plasmid, pED800, of a series of RP1 tra mutants showed that the mutations of all those retaining sensitivity to the P-specific phages PRR1, Pf3, and PR4, or only to PR4, mapped in the Tra1 region, while only 2 out of 20 amber mutations leading to full P-specific phage-resistance did so.     

Tra2betal regulates P19 neuronal differentiation and the splicing of FGF-2R and GluR-B minigenes

The present study demonstrates that the expression of Tra2beta1 (Transformer 2-beta1) proteins, an SR (serine/arginine rich) protein, is developmentally up-regulated in a neural-specific pattern. The up-regulation is also observed in RA (retinoic acid) induced neural differentiation of P19 cells. Tra2betal proteins are located in the nuclei of P19 cells, which are consistent with its functional site as an SR protein. The over-expression of Tra2betal proteins promotes RA induced neuronal differentiation of P19 cells. In P19 cells, the splicing of FGF-2R (fibroblast growth factor receptor 2) minigene produces the BEK form, while the alternative splicing of GluR-B (glutamate receptor subunit B) minigene generates two products, the Flop and the Truncated isoforms. Tra2betal inhibits the BEK splicing, but it promotes the Flop splicing. The results therefore suggest that Tra2betal is involved in the regulation of alternative splicing processes during neural development, peculiarly the splicing of FGF-2R and GluR-B genes. Both FGF-2R and GluR-B genes are known to play important roles in neural differentiation.     

Antisense RNA in Replication Control of the INC FII Plasmid R1

Abstract

This paper gives a short summary of some approaches to investigate antisense RNA control in general, exemplified by studies of a particular model system - replication control of plasmid R1.     

The complete DNA sequence and analysis of R27, a large IncHI plasmid from Salmonella typhi that is temperature sensitive for transfer

Salmonella typhi, the causative agent of typhoid fever, annually infects 16 million people and kills 600 000 world wide. Plasmid-encoded multiple drug resistance in S.typhi is always encoded by plasmids of incompatibility group H (IncH). The complete DNA sequence of the large temperature-sensitive conjugative plasmid R27, the prototype for the IncHI1 family of plasmids, has been compiled and analyzed. This 180 kb plasmid contains 210 open reading frames (ORFs), of which 14 have been previously identified and 56 exhibit similarity to other plasmid and prokaryotic ORFs. A number of insertion elements were found, including the full Tn10 transposon, which carries tetracycline resistance genes. Two transfer regions, Tra1 and Tra2, are present, which are separated by a minimum of 64 kb. Homologs of the DNA-binding proteins TlpA and H-NS that act as temperature-regulated repressors in other systems have been located in R27. Sequence analysis of transfer and replication regions supports a mosaic-like structure for R27. The genes responsible for conjugation and plasmid maintenance have been identified and mechanisms responsible for thermosensitive transfer are discussed.