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.

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.

Bacteriological examination of removed cerebrospinal fluid shunts.

A conventional method of bacteriological examination of removed cerebrospinal fluid shunts was compared with another method which relies on microscopic and cultural examination of intraluminal fluid. Fifty-five shunts were tested. All eight cases of clinical shunt infection gave positive results with the latter method, whereas a further 23 shunts yielded positive cultures by the conventional method in the absence of clinical infection. The consequences of missed infections due to omission of microscopic examination and overdiagnosis using the conventional culture method are discussed.

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.

Chromosome transfer from F-lac+ strains of Escherichia coli K-12 mutant at recA, recB, or recC.

The frequency of chromosome transfer from various recombination-deficient F-lac(+) donor strains was estimated by standardizing the yield of conjugants receiving a male chromosomal marker against the level of episome transfer in the mating mixture. The efficiency of chromosome transfer from newly formed F-lac(+) cells carrying recB21 or recC22 was more than 50% of the wild-type value, although it was about 10 and 20%, respectively, if the male cell lines had become established. In contrast, recA13 donors transmitted the chromosome with less than 10(-4) of the normal frequency. If chromosome transfer from F-lac(+) strains reflects the cutting and subsequent joining of homologous single strands of episomal and chromosomal deoxyribonucleic acid by recombination, these results imply that the completed unions are not made in recA cells, but can be effected with more than 50% of normal efficiency in newly formed partial diploids mutant at either recB or recC. Thus, the defective stage in recA mutants may precede strand joining, whereas the deficiency in recB or recC cells may involve a later step in recombinant formation.

Partial suppression of the phenotype of Escherichia coli K-12 dnaG mutants by some I-like conjugative plasmids.

Three I-like conjugative plasmids, ColIdrd1, R144drd3, and R64drd11, which are derepressed for functions involved in conjugation, were found to suppress at least partially the phenotype of temperature-sensitive dnaG mutants of Escherichia coli K-12, as judged from the kinetics of deoxyribonucleic acid synthesis at elevated temperature in newly formed and established plasmid-containing strains. In contrast, the corresponding wild-type plasmids and three F-like derepressed conjugative plasmids, F101, R100drd1, and R1drd16, all failed to suppress. Suppression is presumably caused by a different plasmid-determined function from that which promotes survival of ultraviolet-irradiated bacteria, because both the wild-type I-like plasmids and their drd mutants protected irradiated bacteria. One possible interpretation of these results is that the product of a gene carried by certain I-like plasmids can substitute for the bacterial dnaG gene product during ongoing deoxyribonucleic acid replication.

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.

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.

Conjugative transfer of IncI1 plasmid DNA primase.

DNA primase of ColIb-P9drd-1 generates RNA primers that are thought to initiate DNA synthesis on the conjugatively transferred strand of the plasmid. To examine whether plasmid-specified primase is transferred during conjugation, we exploited the property of the enzyme to promote bacterial DNA replication in dnaG (primase-defective) mutants of Escherichia coli. It was found that dnaG3 recipient cells, treated with rifampicin to inhibit transcription, recovered ability to synthesise bacterial DNA by a process requiring an active plasmid primase gene in donor cells and a functional conjugation system. A non-transferable primase gene in the donor strain complemented a primase-negative derivative of ColIb-P9drd-1, confirming that the enzyme responsible for recovery was supplied by donor cells. The implication is that certain proteins are transmitted from donor cells to promote conjugative metabolism of plasmid DNA in the recipients.

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.

A novel priming system for conjugal synthesis of an IncI alpha plasmid in recipients.

Synthesis of DNA complementary to the transferred strand of an IncI alpha plasmid has been shown previously to require DNA polymerase III. The possible involvement of the two defined priming proteins of Escherichia coli K12, RNA polymerase and primase, in initiating this conjugal DNA synthesis had been examined. Primase was inactivated using temperature-sensitive dnaG3 mutants and RNA polymerase was inhibited using rifampicin. When these two proteins were simultaneously inactivated in both parental strains, the average recipient synthesised at least one single-stranded equivalent of R144drd-3 before the rifampicin-treated donors lost the ability to transmit DNA. It is proposed that the product of a plasmid transfer gene is responsible for initiating this DNA synthesis in recipients. The results imply that this protein is supplied by the donors.

A colI-specified product, synthesized in newly infected recipients, limits the amount of DNA transferred during conjugation of Escherichia coli K-12.

The amount of ColI DNA transferred between mating cells of Escherichia coli K-12 increased about fourfold when rifampin-resistant donors were mated with sensitive recipients in the presence of the drug. Conjugational synthesis of ColI in dnaB recipients, shown primarily to reflect conversion of the transferred DNA into double-stranded material, was also enhanced when the recipients were treated with either rifampin or streptomycin. It is suggested that the amount of ColI transfer is normally limited by the synthesis of one or more proteins in the newly infected recipients. The protein is thought to be plasmid-specified because rifampin also quadrupled transfer to UV-irradiated recipients which were deficient in the transcription of the resident DNA. Successive strands of ColI appear to be transferred discontinuously, because the transferred DNA accumulated in normal and rifampin-treated recipients in the form of circular and linear monomeric units. Although rifampin treatment of recipients also increased transfer of a second Ialpha plasmid, R144drd-3, by about four times, the drug failed to cause a substantial increase of Flac transfer in comparable matings.

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.

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.

Incidence of postoperative adhesion obstruction following neonatal laparotomy.

Of 649 neonates undergoing laparotomy in a 10 year period, 54 (8.3 per cent) developed adhesion related intestinal obstruction requiring surgical treatment. In 16 infants the obstruction followed a period of prolonged postoperative ileus, while the remaining 38 had completely recovered from the previous surgical procedure before the development of obstruction. The adhesion obstruction occurred after a single neonatal laparotomy in 35 cases but the remaining 19 had undergone subsequent laparotomies; 75 per cent of the obstructions developed within 6 months and 90 per cent within 1 year of surgery. The highest risk groups were infants undergoing correction of gastroschisis (15.4 per cent) and malrotation (15 per cent). There were nine deaths, two of which were a direct consequence of the adhesion obstruction.

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.

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.

Induction of prophage lambda during the division cycle of Escherichia coli.

When synchronous populations of Escherichia coli B/r (lambda) were exposed to low doses of ultraviolet light, the yield of infective centres varied with cell age. The yield was highest if the lysogenic bacteria were irradiated at a time which coincides approximately with the termination of rounds of DNA replication and it was lowest when dividing cells were irradiated. No such variation was detected following either irradiation of excision-defective lysogenic cells or thermal induction of lambdacI857 prophage in irradiated bacteria. It is suggested that the variation reflects a relationship between prophage induction and inhibition of cell division. This hypothesis is supported by data showing that irradiation promoted induction and curtailed division in E. coli K12 dnaA mutants which were dividing in the absence of DNA replication.

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.