The activity of a single-stranded promoter of plasmid ColIb-P9 depends on its secondary structure.

The leading region of the conjugal bacterial plasmid ColIb-P9 contains three dispersed repeats of a 328 bp sequence homologous to Frpo, a sequence from plasmid F that acts as a promoter in single-stranded DNA. One of these sequences, ssi3, inactive in the double-stranded form, promoted in vitro transcription exclusively from the single strand that is transferred during conjugation. Promoter activity was dependent on the presence of RNA polymerase holoenzyme containing sigma 70. Transcription initiated from the position predicted from folding the single-stranded DNA to form a pseudo double-stranded hairpin structure containing recognizable -35 and -10 promoter elements. Footprinting of RNA polymerase holoenzyme on single-stranded ssi3 DNA was consistent with this suggestion. Mutagenesis of the putative -35 region inactivated the promoter, but random mutations in the -10 region had little effect. The putative -10 region is a poor match to the consensus sequence and contains mismatched bases. Elimination of these mismatches invariably destroyed single-strand promoter activity. These observations reveal the crucial contribution of the unpaired bases in the -10 region in potentiating the formation of the productive open complex with RNA polymerase.

DNA-independent transport of plasmid primase protein between bacteria by the I1 conjugation system.

The ColIb-P9 (IncI1)-encoded conjugation system supports transfer of the plasmid T-strand plus hundreds of molecules of the Sog polypeptides determined by the plasmid primase gene. Here, we report that Sog primase is abundantly donated to the recipient cell from cells carrying a non-transferable ColIb plasmid deleted of the nic site essential for DNA export. Such DNA-independent secretion of Sog primase is typical of authentic conjugation, both in being blocked when the recipient cell specifies the entry exclusion function of ColIb and in requiring the thin I1 pilus encoded by the ColIb pil system under the mating conditions used. It is proposed that Sog polypeptides form a complex with the ColIb T-strand during conjugation and aid DNA transport through processive secretion of the proteins into the recipient cell. Functional and genetic relationships between the ColIb conjugation system and other type IV secretion pathways are discussed.

Expression of leading region genes on IncI1 plasmid ColIb-P9: genetic evidence for single-stranded DNA transcription.

The leading region of a plasmid is the first sector to enter the recipient cell in bacterial conjugation. This sector of IncI1 plasmid ColIb-P9 includes genes that are transcribed in a transient pulse early in the conjugatively infected cell to promote establishment of the immigrant plasmid. Evidence is presented that the burst of gene expression is regulated by a process which is independent of a repressor but dependent on the orientation of the genes on the unique plasmid strand transferred in conjugation. The nucleotide sequence of 11.7 kb of the leading region was determined and found to contain 10 ORFs; all are orientated such that the template strand for transcription corresponds to the transferred strand. The leading region contains three dispersed repeats of a sequence homologous to a novel promoter in ssDNA described by H. Masai & K. Arai (1997, Cell 89, 897-907). It is proposed that the repeats are promoters that form in the transferring strand of ColIb to support transient transcription of genes transferred early in conjugation.

Transient transcriptional activation of the Incl1 plasmid anti-restriction gene (ardA) and SOS inhibition gene (psiB) early in conjugating recipient bacteria.

The ardA gene of the enterobacterial plasmid CollbP-9 acts to alleviate restriction of DNA by type I systems, while psiB inhibits induction of the bacterial SOS response. Both genes are transferred early in a round of bacterial conjugation as part of the plasmid leading region. We report here that ardA and psiB are transcribed transiently after their conjugative transport into the recipient cell. Transcript levels, monitored by competitive reverse transcription-polymerase chain reaction (RT-PCR) amplification of RNA templates, started to increase about 5 min after the initiation of conjugation in a cell population and probably before the first round of plasmid transfer was completed. Genetic evidence is given that the expression of ardA and psiB is activated when the genes enter the recipient cell on the transferring plasmid strand. It is proposed that these and other leading region genes function to promote the establishment of the immigrant plasmid in the new host and are expressed by transcription from promoters active only in single-stranded DNA.

IncP plasmids are unusually effective in mediating conjugation of Escherichia coli and Saccharomyces cerevisiae: involvement of the tra2 mating system.

Mobilizable shuttle plasmids containing the origin-of-transfer (oriT) region of plasmids F (IncFI), ColIb-P9 (IncI1), and RP4/RP1 (IncPalpha) were constructed to test the ability of the cognate conjugation system to mediate gene transfer from Escherichia coli to Saccharomyces cerevisiae. Only the Palpha system caused detectable mobilization to yeast, giving peak values of 5 x 10(-5) transconjugants per recipient cell in 30 min. Transfer of the shuttle plasmid required carriage of oriT in cis and the provision in trans of the Palpha Tra1 core and Tra2 core regions. Genes outside the Tra1 core did not increase the mobilization efficiency. All 10 Tra2 core genes (trbB, -C, -D, -E, -F, -G, -H, -I, -J, and -L) required for plasmid transfer to E. coli K-12 were needed for transfer to yeast. To assess whether the mating-pair formation (Mpf) system or DNA-processing apparatus of the Palpha conjugation system is critical in transkingdom transfer, an assay using an IncQ-based shuttle plasmid specifying its own DNA-processing system was devised. RP1 but not ColIb mobilized the construct to yeast, indicating that the Mpf complex determined by the Tra2 core genes plus traF is primarily responsible for the remarkable fertility of the Palpha system in mediating gene transfer from bacteria to eukaryotes.

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.

Distribution of restriction enzyme recognition sequences on broad host range plasmid RP4: molecular and evolutionary implications.

IncP alpha plasmids, exemplified by RP4, are remarkable for their broad host range. They contain strikingly few cleavage sites for many commonly used type II restriction enzymes but an overabundance of sites for certain enzymes that target G + C-rich sequences. To identify factors responsible for these distributions, the recently compiled nucleotide sequence of RP4 was analysed to determine the frequency of tetra- and hexanucleotide motifs in the 49 kb plasmid backbone. This is defined as the sectors encoding basic plasmid functions. The overabundant restriction targets in RP4 are concentrated in the backbone and contain overlapping copies of CGGC/GCCG, identified as the most abundant tetranucleotide motif in the plasmid. Motif frequencies in the RP4 backbone are shown to be similar to those in Pseudomonas aeruginosa, a natural host of RP4, with the notable exception that a number of 6-bp palindromes are underrepresented in the plasmid. It is proposed that 6-bp palindromes were counterselected as type II restriction enzyme recognition sequences. Conjugative transfer of RP4 and R751 (IncP beta) is unusually sensitive to restriction compared to enterobacterial plasmids of the IncFII and IncI1 groups, implying that IncP plasmids experienced particularly strong selection for loss of restriction targets. Pseudomonas spp. of rRNA homology group I specify many type II restriction enzymes that target 6-bp palindromes and are candidates for the evolutionary hosts of IncP alpha plasmids.

Distribution of the ardA family of antirestriction genes on conjugative plasmids.

The ardA gene of I1 plasmid ColIb-P9 was previously shown to alleviate DNA restriction by type I enzymes and to promote conjugative transmission of the unmodified plasmid to a restricting host. To clarify the ecological role of ardA, its distribution was determined on plasmids from 23 incompatibility groups using hybridization to the coding sequence as an assay. Hybridizing sequences, shown by nucleotide sequencing to have at least 60% identity with ardA, were detected on plasmids belonging to the I complex (IncB, I1 and K), the F complex (IncFV) and the IncN group. The ardA homologues were found to specify an antirestriction phenotype which was enhanced by genetic depression of the plasmid transfer system. ardA loci map in plasmid leading regions but show no consistent association with a particular type of origin-of-transfer or a leading region gene of the ssb (single-stranded DNA-binding protein), psiB (plasmid SOS inhibition) and hok (host killing) families. It may be significant that ardA+ plasmids are authentic enterobacterial plasmids and that type I restriction systems are associated historically with members of the Enterobacteriaceae.

DNA processing reactions in bacterial conjugation.

Bacterial conjugation is an important source of genetic plasticity. The initiation complex for conjugative transfer of transmissible plasmids--the relaxosome--is a specific DNA-protein structure that has been isolated from cells and reconstituted from purified components in vitro. Complexes containing uncleaved DNA and DNA cleaved at the nicsite in the origin of transfer (oriT) coexist in equilibrium. Relaxase is usually loaded onto oriT by accessory DNA-binding proteins. Relaxase catalyzes cleavage of a specific phosphodiester bond at nic and becomes covalently linked through a tyrosyl residue to the 5' terminus of the cleaved strand. Cleaved DNA may be unwound for transfer by a plasmid-encoded helicase. Single-strand transfer is thought to occur by a replicative rolling circle mechanism. Termination of a round of transfer is achieved by the cleaving-joining activity of the relaxase linked to the 5' end of the transferring strand. Relationships between DNA processing reactions and conjugative interactions of cell envelopes are particularly obscure aspects of the conjugation cycle.

Evasion of type I and type II DNA restriction systems by IncI1 plasmid CoIIb-P9 during transfer by bacterial conjugation.

Transmission of unmodified plasmid CoIIb-P9 by bacterial conjugation is markedly resistant to restriction compared with transfer by transformation. One process allowing evasion of type I and II restriction systems involves conjugative transfer of multiple copies of the plasmid. A more specialized evasion mechanism requires the Ard (alleviation of restriction of DNA) system encoded by CoIIb. The ard gene is transferred early in conjugation and specifically alleviates DNA restriction by all known families of type I enzyme, including EcoK. CoIIb has no effect on EcoK modification but this activity is impaired by multicopy recombinant plasmids supporting overexpression of ard. Genetic evidence shows that Ard protects CoIIb from EcoK restriction following conjugative transfer and that this protection requires expression of the gene on the immigrant plasmid. It is proposed that carriage of ard facilitates transfer of CoIIb between its natural enterobacterial hosts and that the route of DNA entry is important to the restriction-evasion mechanism.

Zygotic induction of plasmid ssb and psiB genes following conjugative transfer of Incl1 plasmid Collb-P9.

The Incl1 conjugative plasmid Collb-P9 carries a psiB gene that prevents induction of the SOS response in host bacteria. This locus is located 2.5 kb downstream of the ssb (single-stranded DNA-binding protein) gene in the leading region. This portion of Collb is strikingly similar to part of the leading region of the otherwise distinct F plasmid. Expression of psiB and ssb is increased when the host cell is exposed to an SOS-inducing treatment or the Collb transfer system is derepressed. Moreover, expression of both genes on a derepressed plasmid is strongly enhanced in conjugatively infected recipient cells. Carriage of the psiB gene by Collb is shown to prevent a low level of SOS induction following conjugation. Plasmid ssb and psiB genes may function to promote installation of the replicon in the new cell.

Protein transfer into the recipient cell during bacterial conjugation: studies with F and RP4.

Transfer of donor cell proteins to the recipient bacterium was examined in F- and RP4-mediated conjugation. Transfer of a 120 kD polypeptide, identified as the larger product of the plasmid DNA primase gene, was readily detected during RP4-promoted conjugation. The protein was transmitted to the cytoplasm of the recipient, presumably complexed to the transferred ssDNA. F DNA was transferred without detectable association with any cytoplasmic tra protein or with the ssDNA-binding protein encoded by the plasmid. However, a 92 kD protein, possibly F TraD product, was transmitted to the membrane fraction of the recipient cell.

Transfer of tra proteins into the recipient cell during bacterial conjugation mediated by plasmid ColIb-P9.

Selective transfer of the two products of the ColIb primase gene, sog, from donor to recipient cell during conjugation was demonstrated by two independent methods. The transfer of these tra proteins was unidirectional and dependent on DNA transfer. The Sog polypeptides were localized to the cytoplasm of the donor cell, but they appeared to interact with other tra gene products located in the inner membrane. After cell mating, the transferred polypeptides were found to be in the cytoplasm of the recipient cell, and it is estimated that as many as 500 Sog polypeptides were transferred per round of conjugation. It is proposed that these proteins are transferred as a result of an interaction with the single-stranded DNA and that the transferred strand may be coated with Sog polypeptides.

The ssb gene of plasmid ColIb-P9.

The IncI1 plasmid ColIb-P9 was found to carry a single-stranded DNA-binding (SSB) protein gene (ssb) that maps about 11 kilobase pairs from the origin of transfer in the region transferred early during bacterial conjugation. The cloned gene was able to suppress the UV and temperature sensitivity of an ssb-1 strain of Escherichia coli K-12. The nucleotide sequence of the ColIb ssb gene was determined, giving a predicted molecular weight of 19,110 for the SSB protein. Sequence data show that ColIb ssb is very similar to the ssb gene on plasmid F, which is also known to map in the leader region. High-level expression of ssb on ColIb required derepression of the transfer (tra) genes and the activity of the positive regulatory system controlling these genes, suggesting that the SSB protein contributes to the conjugative processing of DNA. A mutant of ColIbdrd-1 carrying a Tn903-derived insertion in ssb was constructed, but it was unaffected in the ability to generate plasmid transconjugants and it was maintained apparently stably in donor cells both following mating and during vegetative growth. Hence, no biological role of ColIb SSB protein was detected. However, unlike the parental plasmid, such ColIb ssb mutants conferred a marked Psi+ (plasmid-mediated SOS inhibition) phenotype on recA441 and recA730 strains, implying a functional relationship between SSB and Psi proteins.

Direction of conjugative transfer of IncI1 plasmid ColIb-P9.

The origin-of-transfer region of ColIb-P9 was inserted into a lambda prophage to give a bacterial chromosome mobilizable by the parental conjugative plasmid. The polarity of mobilization of chromosomal genes indicated that ColIb-P9 transfer is unidirectional, such that the transfer genes adjacent to oriT enter the recipient cell last.

Organization and regulation of the conjugation genes of IncI1 plasmid colIb-P9.

The IncI1 plasmid ColIb-P9 is among a group of related plasmids that encode the I1 type of conjugation system. The I1 system is known to include two morphologically distinct types of pilus, a DNA primase gene (sog) and an exclusion determinant (exc). Transposon mutagenesis and analysis of cloned fragments of ColIb were used to identify the location of these determinants with respect to an EcoRI restriction map. Also identified were the location of the origin of transfer (oriT) and a gene determining an EDTA-resistant nuclease, which is coordinately regulated with the transfer genes. The results indicate that the ColIb tra genes are separated into at least three Tra regions. The pleiotropic nature of transposon insertion mutations in two of these regions suggests that two positive regulators are required for expression of the transfer genes and evidence is also found for a trans-acting repressor. It is suggested that the I1 conjugation system may have evolved following fusion of two distinct types of conjugative plasmid.

Adhesion obstruction following Nissen fundoplication in children.

The incidence of postoperative adhesion intestinal obstruction among 156 children who had undergone Nissen fundoplication for intractable gastro-oesophageal reflux was determined. There were 18 episodes of obstruction in 16 patients (10.3 per cent). The mean interval between fundoplication and adhesion obstruction was 10 months (range 10 days-4 years). Additional procedures performed at the original laparotomy substantially increased the risk of developing obstruction. Relaparotomy for adhesion obstruction was required by 21 per cent of patients who had a Ladd's procedure and 12 per cent who had appendicectomy. Presenting symptoms were not typical of intestinal obstruction because many of these children were unable to vomit. Only three did vomit but all had radiological appearance suggestive of small bowel obstruction. There were two deaths directly related to adhesion obstruction.

Barrett's esophagus in children: a histologic and histochemical study of 11 cases.

Columnar epithelium-lined esophagus is an acquired phenomenon arising secondarily to chronic mucosal injury from gastroesophageal reflux. This report documents 11 children with complications of reflux and the histologic finding of gastric mucosa in the esophagus. Five children had strictures, one requiring esophageal replacement and four treated by antireflux surgery followed by sleeve-resection of a short fibrotic stricture. Specimens from two patients showed mild dysplasia and from six others slight nuclear atypia. Intestinal metaplasia was apparent in one case on routine histology and was revealed in six other cases by mucin histochemical strains. The significance of the histopathologic findings is discussed in the context of possible malignant potential.

Role of sog polypeptides specified by plasmid ColIb-P9 and their transfer between conjugating bacteria.

The sog gene of the conjugative plasmid ColIb-P9 specifies two sequence-related polypeptides with the N-terminal third of the larger product having DNA primase activity. To resolve the function of the C-terminal portion of the polypeptides, we constructed a ColIb mutant containing a Tn5 insertion in the 3' region of sog. The mutation truncated sog gene products without inactivating DNA primase and rendered the plasmid defective in conjugation. Tests for the presence of conjugative pili, for complementation by a sog+ recombinant, and for mobilization of small origin of transfer (oriT) recombinant plasmids indicated that the mutant ColIb allows conjugative aggregation of cells but it is defective in DNA transfer at some stage subsequent to its initiation at oriT. Physical evidence is given that normal sog polypeptides are among a group of proteins transferred selectively from the donor to the recipient cell by a conjugation-specific process. No transfer of the mutant sog proteins was detected. It is proposed that the C-terminal region of sog polypeptides facilitates transfer of single-stranded ColIb DNA between conjugating cells following initiation of transfer at the oriT site, and that in this role the proteins are transmitted to the recipient cell.