A dipeptide insertion in domain I of exotoxin A that impairs receptor binding.

Deletions within the structural exotoxin A gene of 27 or 119 amino acids in domain I of the mature polypeptide, or of 88 or 105 amino acids in domains I and II, resulted in the synthesis of exotoxin A (ETA) polypeptides that were not secreted from Pseudomonas aeruginosa hosts but were localized in the cell membrane. Insertions of a hexanucleotide sequence, either pCGAGCT or pCGAATT, at TaqI sites within the gene resulted in variant exotoxin A polypeptides which were secreted normally. pCGAGCT causes insertion of either Glu-Leu or Ser-Ser in the amino acid sequence of the toxin, while pCGAATT causes insertion of either Glu-Phe or Asn-Ser dipeptides. Although the cytotoxicity of eight variants was unimpaired, that of four others was reduced, and one variant which had a Glu-Phe insert between residues 60 and 61 (ETA-60EF61) was 500-fold less cytotoxic than wild-type exotoxin A. Purified ETA-60EF61 dissociated much faster from mouse LMTK- cells than wild-type ETA, suggesting that the insertion impaired the ability of ETA-60EF61 to interact with exotoxin A receptors. The location of the insert is within a major concavity on the surface of domain I of the exotoxin A molecule, suggesting that this concavity is important for toxin-receptor interaction.

Localization of the control region for expression of exotoxin A in Pseudomonas aeruginosa.

The 2,760-base-pair (bp) PstI-EcoRI segment of the chromosome of Pseudomonas aeruginosa PA103 which carries the exotoxin A structural gene was expressed from an internal promoter when cloned in a pUC9 derivative and transformed into a nontoxigenic mutant of P. aeruginosa PAO1. The unique terminal EcoRI site was deleted, and a new EcoRI site was substituted for a PvuI site located 107 bp 5' to the transcription initiation site. Following EcoRI cleavage, Bal31 deletions were generated from this site, and an EcoRI linker sequence, GGAATTCC, was inserted in place of the deleted DNA. Mutants with deletions located 73 bp or more upstream of the transcription initiation site retained normal expression, whereas in mutants with deletions extending into the region 69 bp or less upstream of this site, exotoxin synthesis was greatly reduced. From a KpnI site located 473 bp 3' to the transcription initiation site, a similar series of Bal31 deletion mutants were generated in which the inserted EcoRI linker sequence was located within the same 72-bp region. Pairs of mutants from the two deletion series were identified in which the EcoRI linker was located at the same sequence, and these mutant pairs were ligated to derive a series of constructs in which the EcoRI linker sequence GGAATTCC was substituted for an 8-bp sequence within the 72-bp region. Some of these linker-substituted mutants showed greatly reduced exotoxin A synthesis. The results are consistent with a binding site for a positive activator contiguous with the binding site for an RNA polymerase.

Transcription and expression of the exotoxin A gene of Pseudomonas aeruginosa.

The exotoxin A genes from Pseudomonas aeruginosa strains PA103 and PAO1 have been independently cloned in a pUC9-derived plasmid. In a non-toxigenic mutant of PAO1 as host, the cloned genes directed the synthesis of intact exotoxin A that expressed ADP-ribosyltransferase activity upon treatment with urea and dithiothreitol. Western-blot analysis of culture supernatants identified a polypeptide of 67 kDa, the molecular mass of intact exotoxin A. There was an approximately 15-fold increase in the toxin yield from P. aeruginosa cells carrying a cloned PA103 gene compared to PA103, and a 40-fold increase in the yield of toxin gene yielded about four times more toxin than those carrying the cloned PAO1 gene. Toxin expression was correlated with the presence of a transcript that was initiated 88 bp upstream from the translational start site. Little or no messenger RNA from either cloned gene could be detected in an Escherichia coli host, or in a P. aeruginosa host grown in the presence of 0.1 mM-Fe2+, a condition that inhibits toxin expression. The nucleotide sequences of two regions, each of approximately 500 bp, near the 5' and 3' termini of the structural gene were established. In these regions, three exotoxin A gene from PAO1 has ten base-pair differences compared to the PA103 gene, three in the non-coding region, and seven in the structural gene, four of which should lead to amino-acid differences. No apparent sequence similarities were found between the inferred promoter region of the exotoxin A gene and that of other Pseudomonas genes, nor with the consensus sequence of E. coli promoters.

Nucleotide sequence comparisons of plasmids pHD131, pJB1, pFA3, and pFA7 and beta-lactamase expression in Escherichia coli, Haemophilus influenzae, and Neisseria gonorrhoeae.

The sites of initiation for beta-lactamase mRNA transcription and the nucleotide sequences of beta-lactamase plasmids derived from Haemophilus and Neisseria species were determined. In N. gonorrhoeae, transcription from plasmid pFA3 was initiated from two sites, one located about 20 base pairs (bp) and the other 210 bp upstream of the beta-lactamase initiating codon, whereas in H. influenzae, transcriptional initiation from plasmid pHD131 occurred at two different sites, approximately 150 and 170 bp upstream of the initiating codon. When these plasmids were transformed into Escherichia coli, transcription was initiated at the 150- and 170-bp upstream sites in both plasmids. The nucleotide sequences of both plasmids within the noncoding region upstream of the transcriptional initiation site were identical and, except at two or three nucleotide positions, the sequences were also identical to the corresponding region of Tn3. At one of these positions there is a TA for CG substitution, which correlates in E. coli and Haemophilus sp. with the presence of two strong, overlapping beta-lactamase promoters, initiating transcription at the 150- and 170-bp upstream sites. Over a larger (875-bp) segment comprising most of the sequences of the tnpR and bla genes, the nucleotide sequences of both plasmids were also identical, and although this sequence differed from the corresponding Tn3 sequence at 18 sites, it was identical to that of Tn2, except at one site. The sequence of a second Haemophilus plasmid, pJB1, was identical to that of pHD131 in the same region, except at two nucleotides. All three plasmids were identical in nucleotide sequence in other TnA regions, as well as in regions flanking the TnA sequence, except that the Neisseria plasmid lacked a TnA segment of 3,298 bp [comprising the IR(L) and proximal sequences] together with approximately 273 bp of the non-TnA region adjacent to IR(L). The sequence of a second N. gonorrhoeae plasmid, pFA7, was identical to pFA3, except that the terminal, 3,299 TnA nucleotides were missing.

Variations between the nucleotide sequences of Tn1, Tn2, and Tn3 and expression of beta-lactamase in Pseudomonas aeruginosa and Escherichia coli.

Using S1 nuclease assays, we located the sites of initiation of transcription of the beta-lactamase gene on Tn1 and Tn2. Transcription in Tn2, like that in Tn3, occurred from the P3 promoter, whereas transcription in Tn1 was initiated by two stronger and overlapping promoters, Pa and Pb. The nucleotide sequences of Tn1 and Tn2 were determined over a 1,195-base-pair segment constituting most of the sequences of the tnpR and bla genes and the intervening region. There were six base-pair differences between Tn1 and Tn3. One in the bla regulatory region accounted for the presence of the Pa and Pb promoters, and another in the bla structural gene is consistent with the isoelectric focusing difference found between the Tn1 and Tn3 enzymes. In contrast, there were 24 base-pair differences between Tn2 and Tn3, most of them clustered in one segment of the tnpR gene.

Cloning and expression of a gene segment encoding the enzymatic moiety of Pseudomonas aeruginosa exotoxin A.

Using the broad-host-range plasmid vector pRO1614, we cloned a segment of the gene from Pseudomonas aeruginosa PA103 encoding the enzymatically active part of the exotoxin A protein. Expression of the cloned gene segment has been achieved both in Escherichia coli and in a nontoxigenic P. aeruginosa host, as assayed by the production of exotoxin A-related antigen and by the ability of the gene product to ADP-ribosylate elongation factor 2. Western blot hybridization analysis revealed a series of polypeptides antigenically related to exotoxin A, the largest of which had a molecular weight of ca. 50,000.

Two improved promoter sequences for the beta-lactamase expression arising from a single base-pair substitution.

A mutation in the transposon Tn2660 derived from the plasmid R6K, and resulting in an approximate tenfold increase in penicillin resistance is shown to be a single GC to AT substitution 177 base pairs 'upstream' of the initiation codon of the structural beta-lactamase (bla) gene. This substitution leads to the transcription of two new mRNAs which can be ascribed to the creation of two new overlapping promoter sequences. All the sequences (450bp) examined in the wild-type Tn2660 are identical to those reported in Tn3.

Absence of direct repeats flanking transposons resulting from intramolecular transposition.

Tn2660 is an ampicillin-resistance-conferring transposon with a high degree of homology for the transposon Tn3. The nucleotide sequences flanking the termini of Tn2660 have been determined on plasmids inferred to have resulted from both inter- and intramolecular transposition of Tn2660. In all cases, transposition of Tn2660, as of Tn3, creates 5-bp flanking direct repeats, except following intramolecular transposition resulting from trans ligation. In this case, in R6K replicons, the nucleotide sequence between the two Tn2660 elements is stably inverted from the normal orientation, and 5-bp direct repeats do not flank each transposon, but instead flank opposite ends of the two transposon copies.

Intermolecular and intramolecular transposition and transposition immunity in Tn3 and Tn2660.

Intermolecular transposition of Tn2660 into pCR1 was measured at 30 degrees C in recA- and recA+ hosts as between 2.6 and 5.5 X 10(-3), a similar value to that previously found for Tn3. No cointegrate structures were found under conditions where 10(4) transposition events occurred. Immunity to intermolecular transposition of Tn2660, similar to that found for Tn3 was demonstrated by showing that the above transposition frequency was reduced by a factor of between 10(-3) and 10(-4) when a mutant Tn2660 (resulting in the synthesis of a temperature-sensitive beta-lactamase) was present in the recipient plasmid. Intramolecular transposition of Tn3 was found to occur under the same conditions as previously demonstrated for Tn2660 giving rise to similar end products, in which the newly introduced Tn3 is oriented inversely to the resident Tn3 and the DNA sequence between the two transposons has been inverted. Thus, in all respects functional identity of the transposition activities of Tn3 and Tn2660 is shown, thereby identifying characteristics of intramolecular transposition that are not readily accommodated by current models of transposition.

Recombination between two TnA transposon sequences oriented as inverse repeats is found less frequently than between direct repeats.

Inverse repeats of the transposon Tn2660 in either a ColE1 or an R6K replicon, with or without inversions of the parental DNA sequences between the repeats, show no detectable (less than 2%) evidence of recombination between the repeats after 60 generations of growth in either recA or RecA+ hosts. In contrast, attempts made to construct plasmids which carry two direct repeats by in vitro cleavage and ligation in the recA host were unsuccessful, although homologous plasmids with inverse repeats could be constructed, and other plasmids were found consistent with products of recombination between the direct repeats of the transient intermediate structure. It is concluded that in recA or recA+ hosts recombination between direct repeats of a transposon is frequent, whereas recombination between inverse repeats of a homologous structure has not been observed. A model to explain this difference depends upon a mechanisms that produces a nick in only one of the pair of strands at the internal resolution site (IRS) sequence of the transposon.

Intramolecular transposition and inversion in plasmid R6K.

Selection was made in Escherichia coli K-12 recA hosts carrying plasmid R6K for ampicillin hyperresistance. Twenty-two selected strains were found to carry mutant plasmids, which, from electron microscopy and restriction enzyme analysis, were concluded to arise by a duplication of transposon Tn2660, which confers ampicillin resistance, in all cases the duplicate transposon being in an inverted orientation with respect to the resident Tn2660. A mutant of R6K, pSJC301, which was temperature sensitive for ampicillin resistance was produced by in vitro hydroxylamine treatment of R6K deoxyribonucleic acid. A plasmid hybrid, pSJC102, was constructed by cloning the EcoRI R6K fragment carrying the wild-type beta-lactamase gene into the EcoRI site of ColE1. pSJC301 and pSJC102 were transformed into the same recA host strain to form a stable biplasmid strain. Ampicillin-hyperresistant mutants were selected from this strain and screened for plasmids with a duplication of transposon Tn2660, which occurred with equal frequency in either pSJC301 or pSJC102; of 12 characterized, all were inverse repeats of the resident transposon. All six Tn2660 inserts into pSJC301 determined temperature-sensitive ampicillin resistance, and all six inserts into pSJC102 determined wild-type ampicillin resistance, from which it was inferred that transposition of a duplicate Tn2660 occurs predominantly as an intramolecular event, at least in the multicopy R6K plasmid. In all 28 insertion mutants of R6K, there was an inversion of the deoxyribonucleic acid between the two transposons, whereas in only one of six insertion mutants of pSJC102, inversion had occurred. These results are discussed in terms of current models of transposition.

Physical characterization of plasmids determining synthesis of a microcin which inhibits methionine synthesis in Escherichia coli.

Plasmid deoxyribonucleic acid (DNA) isolated from each of three antibiotic-resistant clinical strains of Escherichia coli producing the same microcin showed multiple bands upon agarose gel electrophoresis. Transformants selected either for microcin resistance or ampicillin resistance yielded plasmid DNA corresponding in size to only one of the multiple bands. Plasmids, isolated from all three hosts, which determined microcin resistance and microcin production measured about 4 megadaltons by sucrose density, restriction enzyme, and contour length analyses; cleavage of the DNAs by each of eight restriction enzymes showed the same response, and DNA-DNA hybridization indicated complete homology. The antibiotic resistance plasmids of the three host strains were uniformly larger, were of different sizes, and showed different restriction enzyme cleavage patterns. One of these R plasmids (pCP106) also determined the synthesis of the same microcin, and DNA-DNA hybridization studies indicated an approximate 2.4-megadalton homology with the 4-megadalton microcin plasmid pCP101. The microcin plasmids were present at approximately 20 copies per genome equivalent and were nonconjugative, whereas the R plasmids had a copy number of about 1, were conjugative, and could mobilize the microcin plasmid. Microcin plasmid pCP101 showed replication properties similar to those of a number of small multicopy plasmids such as ColE1.

Transposition of a duplicate antibiotic resistance gene and generation of deletions in plasmid R6K.

Transformation experiments showed that spontaneous deletions which result in loss of streptomycin resistance and an increase in conjugal transfer efficiency are present at a frequency of about 10(-4) in plasmid molecules of R6K. Similar deletions were thus readily selected by conjugal transfer of R6K, and their appearance was dependent upon recA+ activity in either donor or recipient host. The deoxyribonucleic acid segment deleted in four mutants examined was concluded to extend from the same terminus of the transposon, TnA, in the same direction, but to different extents, and to retain the TnA region intact. Insertions of a duplicate TnA element were found in R6K plasmids isolated from strains selected for increased ampicillin resistance, which were unstable in recA+ strains. In four plasmids examined after transfer to a recA host, an inverted repeat of the preexisting TnA element was shown to have been inserted at a similar location and was in two instances associated with deletions which extended from the same direction as those described above. The deletions are ascribed to the result of recA+-dependent recombination between direct repeats of TnA.

Molecular structure of an R factor, its component drug-resistance determinants and transfer factor.

Plasmid DNA from Escherichia coli strains harboring drug resistance either of the infectious or noninfectious kind has been separated by CsCl centrifugation of crude cell lysates in the presence of ethidium bromide and examined by electron microscopy. Plasmid deoxyribonucleic acid (DNA) from an S(+) strain (which has the property of noninfectious streptomycin-sulfonamide resistance) consists of a monomolecular covalently closed circular species of 2.7 mum in contour length (5.6 x 10(6) atomic mass units; amu). DNA from a strain carrying a transfer factor, termed Delta, but no determinant for drug resistance, is a monomolecular covalently closed circular species of 29.3 mum in contour length (61 x 10(6) amu). DNA from either Delta(+)A(+) or Delta(+)S(+) strains, (which are respectively ampicillin or streptomycin-sulfonamide resistant, and can transfer this drug resistance), shows a bimodal distribution of molecules of contour lengths 2.7 mum and 29.3 mum, whereas DNA from a (Delta-T)(+) strain (showing infectious tetracycline resistance) contains only one species of molecule measuring 32.3 mum (67 x 10(6) amu). We conclude that ampicillin resistance is carried by a DNA molecule (the A determinant) of 2.7 mum, and streptomycin-sulfonamide resistance is carried by an independent molecule (the S determinant) of similar size. These molecules are not able to effect their own transfer, but can be transmitted to other cells due to the simultaneous presence of the transfer factor, Delta, which also constitutes an independent molecule, of size 29.3 mum. In general, there appears to be little recombination or integration of the A or S molecules into that of Delta, although a small proportion (5-10%) of recombinant molecules cannot be excluded. In contrast, the third drug-resistance determinant, that for tetracycline resistance (denoted as T), is integrated in the Delta molecule to form the composite structure Delta-T of size 32.3 mum, which determines infectious tetracycline resistance. The Delta(+)A(+) and Delta(+)S(+) strains are defined as harboring plasmid aggregates, and the (Delta-T)(+) strain is defined as carrying a plasmid cointegrate; the properties of all three strains are characteristic of strains harboring R factors. These results are compatible with the previously published genetic data. The number of Delta molecules per cell appears to be equal to the chromosomal number irrespective of growth phase, and this plasmid can thus be defined as stringently regulated in DNA replication. In contrast, S and A exist as multiple copies, probably in at least a 10-fold excess of chromosomal copy number. S and A can thus be defined as relaxed in the regulation of their DNA replication.