Molecular characterization of a small Haemophilus influenzae plasmid specifying beta-lactamase and its relationship to R factors from Neisseria gonorrhoeae.

The ampicillin-resistant Haemophilus influenzae strain Ve445 which caused purulent meningitis and septicaemia in a newborn child in Germany contained a 4.4 megadalton (Mdal) plasmid (pVe445) and produced a TEM type beta-lactamase. The transformation to ampicillin resistance of a sensitive Escherichia coli strain with isolated pVe445 DNA proved that the structural gene for the beta-lactamase resided on this plasmid genome. Molecular DNA-DNA hybridization studies and electron microscope DNA heteroduplex analysis indicated that pVe445 probably contained 38 to 41% of the ampicillin translocation DNA segment (TnA) found on R factors of enteric origin. The TnA fragment present in pVe445 most likely does not contain both of the inverted repeat sequences of TnA. DNA-DNA polynucleotide sequence studies indicated that the 4.4 Mdal plasmid pVe445 was unrelated to the 30 to 38 Mdal H. influenzae R plasmids but was closely related to the 4.1 Mdal ampicillin resistance specifying H. influenzae plasmid RSF0885 isolated in the U.S.A. The H. influenzae plasmid pVe445 shared 91% of its base sequences with the beta-lactamase specifying Neisseria gonorrhoeae plasmid pMR0360 (4.4 Mdal) and had 85% of its base sequences in common with the beta-lactamase specifying N. gonorrhoeae plasmid pMR0200 (3.2 Mdal). All of the four 3.2 to 4.4 Mdal beta-lactamase specifying R plasmids of H. influenzae and N. gonorrhoeae investigated probably have a common evolutionary origin.     

Relationships among some R plasmids found in Haemophilus influenzae.

Tetracycline resistance in a strain of Haemophilus influenzae isolated in the United Kingdom was found to be determined by an apparently non-selftransmissible plasmid of 31 X 10(6) daltons (31 MDal), designated pUB701. Deoxyribonucleic acid hybridization studies indicated that pUB701 shares about 70% base sequence homology with the 30-MDal ampicillin resistance R plasmid RSF007 isolated in the United States from H. influenzae, and 64% sequence homology with the 38-MDal tetracycline and chloramphenicol resistance R plasmid pRI234, isolated in the Netherlands. Heteroduplex studies between RSF007 and pUB701 confirmed the fact that these plasmids were largely homologous, except that pUB701 contained the tetracycline resistance transposon TnD, whereas RSF007 contained the ampicillin resistance transposon TnA. A strain of H. parainfluenzae resistant to both chloramphenicol and tetracycline carried two species of plasmid deoxyribonucleic acid of 2.7 and 0.75 MDal. We were unable to prove that either resistance was plasmid-borne in this strain. Hybridization studies with a [3H]thymine-labeled tetracycline resistance enteric plasmid suggested that the tetracycline transposon was integrated into the chromosome of H. parainfluenzae UB2832. We conclude either that the strains we studied received R factors of the same incompatibility group bearing different resistance genes, or that different resistance genes were translocated to a commom resident plasmid of H. influenzae.     

Molecular nature of a plasmid specifying beta-lactamase production in Haemophilus ducreyi

We characterized pJB1, the plasmid previously reported to mediate beta-lactamase production in Haemophilus ducreyi. We studied its relationship to pMR0360 and RSF0885, the plasmids responsible for beta-lactamase production in Neisseria gonorrhoeae and Haemophilus parainfluenzae, respectively. Although pJB1 was maintained as a multicopy pool in Escherichia coli, it was not stably maintained in the absence of antibiotic selection. Electron microscope heteroduplex studies showed that it carried 100% of the transposable ampicillin resistance sequence TnA. This sequence was transposed to plasmid pUB307 at a low rate. Heteroduplexes between pMR0360 and pJB1 showed that they contained 3.3 megadaltons of homologous sequences. Two sets of nonhomologous sequences, one a TnA sequence and the other a non-TnA sequence, took the form of insertion loops. For plasmids pMR0360 and RSF0885, previously shown to be highly related, the nonhomologous sequences took the form of a substitution loop. We concluded that all three plasmids shared major portions of their sequences but differed in discrete segments. pJB1 was the first such plasmid to have a physically and functionally intact TnA sequence.     

Transformation of Haemophilus influenzae by plasmid RSF0885.

Plasmid RSF0885, which conferred ampicillin resistance, transformed competent Haemophilus influenzae cells with low efficiency (maximum, less than 0.01%). As judged by competition experiments and uptake of radioactivity, plasmid RSF0885 deoxyribonucleic acid was taken up into competent H. influenzae cells several orders of magnitude less efficiently than H. influenzae chromosomal deoxyribonucleic acid. Plasmid RSF0885 transformed cells with even lower efficiency than could be accounted for by the low uptake. Transformation was not affected by rec-1 and rec-2 mutations in the recipient, and strains cured of the plasmid did not show increased transformation. Plasmid molecules cut once with a restriction enzyme that made blunt ends did not transform. Transformation was favored by the closed circular form of the plasmid.     

Molecular characterization of two beta-lactamase-specifying plasmids isolated from Neisseria gonorrhoeae.

The molecular nature of two distinct gonococcal R plasmids, 4.4 X 10(6) and 3.2 X 10(6) daltons, encoding beta-lactamase activity were examined. Both plasmids contained about 40% of the transposable ampicillin resistance sequence Tn2. Deoxyribonucleic acid-deoxyribonucleic acid polynucleotide sequence studies have shown that the two gonococcal plasmids share about 70% of their sequences and are closely related to RSF0885, a 4.1 X 10(6)-dalton plasmid found in a beta-lactamase-producing strain of Haemophilus influenzae. All three of these R plasmids possess a guanine-plus-cytosine content of 0.40 to 0.41 mol fraction and are present as multicopy gene pools in their bacterial hosts.     

Intraspecific and intergeneric mobilization of non-conjugative resistance plasmids by a 24.5 megadalton conjugative plasmid of Neisseria gonorrhoeae

pLE2451, a 24.5 megadalton conjugative plasmid from Neisseria gonorrhoeae, was capable of efficiently mobilizing gonococcal beta-lactamase plasmids between gonococci and from gonococci to Haemophilus influenzae and restriction-deficient Escherichia coli. Donor strains of N. gonorrhoeae carrying pLE2451 were also found to be capable of mobilizing a variety of non-conjugative plasmids originally derived from enteric bacteria or Haemophilus species when such plasmids were resident in E. coli. Nevertheless, pLE2451 was not detected physically in E. coli or H. influenzae transconjugants. This suggests that the plasmid is unstable in these hosts but survives transiently to provide transfer functions for mobilization. The proficiency of pLE2451 in promoting intraspecific and intergeneric mobilization was not paralleled by pUB701, pRI234 and pFR16017, a series of conjugative plasmids derived originally from Haemophilus species. These plasmids were incapable of mobilizing even Haemophilus beta-lactamase plasmids, such as RSF0885, between Haemophilus species.     

Uptake of plasmid deoxyribonucleic acid by Haemophilus

The uptake of circular and linear plasmid RSF0885 deoxyribonucleic acids, (DNAs) obtained from Haemophilus parainfluenzae 14, in both homologous and heterologous recipients was studied and compared with that of chromosomal DNA. High concentrations of divalent cations stimulated the uptake of either circular or linear plasmid DNA in H. parainfluenzae 14 competent cells but did not affect the uptake of chromosomal DNA. The biological activity of linear plasmid DNA was similar to that of circular DNA, and the transforming efficiencies for ampicillin resistance of both molecular forms were stimulated by divalent ions. Plasmid DNA was taken up efficiently either with or without the addition of divalent ions but was not biologically active in the heterologous Haemophilus influenzae Rd recipient. Our results suggest that in H. parainfluenzae 14 some of the steps for chromosomal and plasmid DNA uptake are different.     

New shuttle vectors for Haemophilus influenzae and Escherichia coli: P15A-derived plasmids replicate in H. influenzae Rd

With the aim of identifying new plasmids useful for molecular cloning in Haemophilus influenzae, several P15A-derived plasmids were tested for their ability to transform H. influenzae Rd. The four plasmids tested, pACYC177, pACYC184, pSU2718, and pSU2719 were all able to establish in H. influenzae Rd. The plasmids were stable, could be purified by standard protocols, and were compatible with a plasmid carrying the RSF0885 origin of replication.     

Mobilization of nonconjugative antibiotic resistance plasmids in Haemophilus ducreyi.

A clinical isolate of Haemophilus ducreyi was found to harbor three plasmids: a 23.5-megadalton (Mdal) phenotypically cryptic plasmid, a 7.0-Mdal ampicillin resistance plasmid, and a 4.0-Mdal sulfonamide resistance plasmid. The two smaller plasmids were transferable by conjugation to Haemophilus recipients, but only if the donor cell harbored the 23.5-Mdal plasmid as well, indicating that this large plasmid had mobilizing capabilities. Transfer was also possible to Escherichia coli recipients. Haemophilus influenzae transconjugants which had acquired both the 23.5-Mdal plasmid and one of the R-plasmids could subsequently retransfer the R-plasmid to other Haemophilus recipients at higher frequencies. A derivative of the 23.5 Mdal plasmid was isolated which was shown by restriction endonuclease analysis to contain an ampicillin resistance transposon and to have retained its conjugative ability.     

Transposition of a plasmid deoxyribonucleic acid sequence that mediates ampicillin resistance: identity of laboratory-constructed plasmids and clinical isolates.

The structural gene for ampicillin resistance resides upon a 3.2 X 10(6)-dalton sequence of deoxyribonucleic acid, TnA that can be transposed from replicon to replicon in laboratory experiments. TnA was transposed from a large conjugative plasmid to a small nonconjugative plasmid, RSF1010. Several RSF1010::TnA plasmids isolated in these laboratory experiments have been shown to be identical to plasmids found in clinical isolates. These data provide direct support to the theory that transposition of drug resistance genes play a key role in the evolution of R plasmids.     

Transformation of Haemophilus influenzae by plasmid RSF0885 containing a cloned segment of chromosomal deoxyribonucleic acid.

A plasmid containing a single cloned insertion of Haemophilus influenzae chromosomal deoxyribonucleic acid that carried a novobiocin resistance marker was 2.6 times larger than the parent plasmid, RSF0885, which conferred ampicillin resistance. The most frequent type of transformation by this plasmid (designated pNov1) was the transfer of novobiocin resistance to the chromosome, with the loss of the plasmid from the recipient. In accord with this observation, after radioactively labeled pNov1 entered a competent cell, it lost acid-insoluble counts, as well as biological activity. The level of ampicillin transformation, which involved establishment of the plasmid, was almost two orders of magnitude lower than the level of novobiocin transformation. Both types of transformation were depressed profoundly in rec-1 and rec-2 mutants. Ampicillin transformants of wild-type cells always contained plasmids that were the same size as pNov1, although most of these transformants were not novobiocin resistant. Plasmid pNov1 in wild-type cells but not in rec-1 or rec-2 cells often recombined with the chromosome, causing a homologous region of the chromosome to be substituted for part of the plasmid, as shown by restriction and genetic analyses. Our data suggested that plasmid-chromosome recombination took place only around the time when the plasmid entered a cell, rather than after it became established.     

TnA-directed deletion of the trp operon from RSF2124-trp in Escherichia coli

Plasmid pMT-trp was constructed by digestion of RSF2124-trp with restriction endonuclease PstI and ligation with T4 ligase. In pMT-trp about 78% of the DNA of transposon TnA from RSF2124-trp was deleted, and hence the gene for ampicillin resistance was lost. All Trp- segregants from pMT-trp carriers in Escherichia coli W3110 and its derivatives were found to have lost the entire plasmid. On the other hand, deletion plasmids which had lost the trp operon were found among Trp- segregants from RSF2124-trp carriers, particularly from the mutant strain trpAE1 trpR tnaA. The experimental fact that deletion occurred exclusively in RSF2124-trp suggests that the presence of TnA in the plasmid (RSF2124-trp) was responsible for the deletion.     

Origin of Haemophilus influenzae R factors.

The Haemophilus influenzae R plasmids specifying resistance against one, two, or three antibiotics which have emerged in different parts of the world were shown to have closely related but not identical plasmid cores. The gene for ampicillin resistance in the H. influenzae plasmid pKRE5367 is part of a transposon similar to Tn3, which was transposed from pKRE5367 onto RSF1010 in Escherichia coli. An indigenous H. influenzae plasmid (pW266) was isolated. Its properties correspond to those of the H. influenzae R plasmids, except for the presence of a drug resistance transposon. The in vitro-generated H. influenzae R plasmids carrying an ampicillin resistance transposon, a tetracycline resistance transposon, and a transposon for combined tetracycline-chloramphenicol resistance resembled the natural isolates. The findings support the hypothesis that the R plasmids of H. influenzae are of multiclonal evolutionary origin.     

Molecular nature of two Haemophilus influenzae R factors containing resistances and the multiple integration of drug resistance transposons.

The 36-megadalton Haemophilus influenzae R plasmid pHK539 was found to specify resistance to tetracycline (Tc) and ampicillin (Ap). It was shown by molecular hybridization studies and by electron microscopy that the plasmid pHK539 contained the tetracycline translocation deoxyribonucleic acid (DNA)segment (TnTc) as well as the ampicillin translocation segment (TnAp). The TnAp was integrated in the stem of TnTc. The 34-megadalton H. influenzae R plasmid pRI234 carried a translocatable DNA segment which specified both tetracycline and chloramphenicol (Cm) resistance. Self-annealing and DNA-DNA heteroduplex experiments indicated that this transposon is probably composed of TnTc containing an insertion of a chloramphenicol resistance transposon (TnCm). TnCm is inserted into one of the components of the TnTc inverted repetitions and is itself flanked on both sides by long inverted repetitions. The H. influenzae plasmids pHK539 and pRI234 had more than 60% of their polynucleotide sequences in common with all the other 30- to 40-megadalton R factors recently found in H. influenzae isolates from different countries. The tetracycline-chloramphenicol resistance transposon of pRI234 was integrated twice at different sites in the plasmid after its growth in medium containing tetracycline. The presence of the two copies of the transposon was correlated with higher minimum inhibitory concentrations against tetracycline as well as against chloramphenicol. After its growth in medium containing tetracycline, the H. influenzae R plasmid pFR16017 specifying Tc resistance contained one, two, three, or even four copies of TnTc integrated at different sites in the plasmid, or the loop of TnTc was amplified. The heterogeneity of the pFR16017 plasmid was seen in all single-colony isolates and correlated with a higher minimum inhibitory concentration against tetracycline.     

Genetic transformation in bacteria

Certain species of bacteria can become competent to take up high molecular weight DNA from the surrounding medium. DNA homologous to resident chromosomal DNA is transported, processed and recombined with the resident DNA. There are some variations in steps leading to transformation between Gram-positive bacteria likebiplococcus pneumoniae and Gram-negative bacteria represented byHaemophilus influenzae but the integration is by single-strand displacement in both cases. Plasmid (RSF0885) transformation is low inHaemophilus influenzae but this is increased significantly if (homologous) chromosomal DNA is spliced to plasmid DNA. InHaemophilus influenzae, rec1 function is required for peak transformation with chimeric plasmids. Chimeric plasmid fixed presumably extrachromosomally undergoes frequent recombination between homologous segments contained in resident chromosome and the plasmid.     

Plasmid transfer in Haemophilus influenzae.

Twenty-nine strains of Haemophilus influenzae highly resistant to ampicillin, chloramphenicol, or tetracycline were examined for the presence of plasmids. Agarose gel electrophoresis of ethanol-precipitated cell extracts revealed large plasmids in 11 strains, of which 7 were conjugative. Plasmid transfer by conjugation between isogenic strains was quite efficient, but transfer between different serotypes was nearly always much more inefficient. Type I or II restriction enzymes do not appear to be barriers to this transfer. Encapsulated cells can be both efficient donors and recipients. Small plasmids were seen in three strains, but only two of the three are resistance factors (RSF0885, pUB703). Thus, in 17 isolates antibiotic resistance genes are believed to be located in the bacterial chromosome. Most of these resistances could be transferred by genetic transformation into the widely used Rd strain. In some cases transfer of chromosomal resistance into conjugative plasmids was observed in both rec+ and rec host cells. Since transfer by conjugation seems to be the more efficient process, it is puzzling that in the majority of the 29 isolates studied resistance genes appeared to be in the chromosome.     

Molecular properties of transmissible R factors of Haemophilus influenzae determing tetracycline resistance.

The tetracycline-resistant Haemophilus influenzae strains LU121 and FR16017, recently isolated in West Germany, each harbour a plasmid; that of the former (pLU12U) has a mol. wt of 31.5 X 10(6) and that of the latter (pFR16017) has a mol. wt of 33 X 10(6). Conjugation and DNA-DNA hybridization studies have shown that both plasmids are self-transmissible and carry tetracycline-resistance genes. The purified plasmid DNA of H. influenzae strain LU121 transformed a sensitive Escherichia coli strain to tetracycline resistance. The two R factors are closely related to the H. influenzae plasmid specifying ampicillin resistance (pKRE5367). Electron microscope DNA heteroduplex analysis indicated that pLU121 and pFR15017 probably carry the tetracycline-resistance transposon TnD and that pKRE5367 probably carries the ampicillin-resistance transposon TnA. There is more than one integration site for the insertion which probably represents TnD in pFR15017. All three plasmids have a similar plasmid core and could have a common evolutionary origin.     

Origin of small beta-lactamase-specifying plasmids in Haemophilus species and Neisseria gonorrhoeae.

Fifty-nine percent of unselected strains of Haemophilus parainfluenzae were found to carry small, phenotypically cryptic plasmid DNA species. Using filter blot hybridization, we found several plasmids which were homologous to the small beta-lactamase-specifying plasmids pJB1 and pFA7, which were originally isolated from Haemophilus ducreyi and Neisseria gonorrhoeae, respectively. Detailed filter hybridization studies combined with electron microscope heteroduplex analysis suggested that three cryptic plasmids are completely homologous to the non-TnA sequences of pJB1. One cryptic plasmid was found to be highly homologous to pJB603, a small beta-lactamase plasmid previously found in two isolates of H. influenzae. A second group of plasmids were found to carry sequences homologous to pJB1 and other sequences homologous to pJB603. These results strongly suggest that small beta-lactamase plasmids found in Haemophilus species and N. gonorrhoeae may have arisen by insertion of the transposable beta-lactamase-specifying element TnA into small, phenotypically cryptic replicons resident in H. parainfluenzae. Attempts to reproduce such a recombination event in the laboratory were not successful.