Lack of expression of RP4-specified beta-lactamase in Azospirillum brasilense

Plasmid RP4, which normally confers resistance to ampicillin (Apr), tetracycline (Tcr), and kanamycin (Kmr) to its hosts, failed to express enhanced Apr when transferred from Escherichia coli to Azospirillum brasilense which has its own intrinsic beta-lactamase. Even in a beta-lactamase-deficient mutant, A. brasilense RG-D16, no increase in beta-lactamase or significant Apr appeared following transfer of RP4. However, A. brasilense RG (RP4) and A. brasilense RG-D16 (RP4) did exhibit Tcr Kmr. When RP4 was transferred back from A. brasilense to E. coli all three drug resistances and beta-lactamase activity were fully expressed.     

Transformation and Physical Properties of R-Factor RP4 Transferred from Escherichia coli to Rhizobium trifolii

Transformation of R-factor RP4 specifying resistance to ampicillin, kanamycin, and tetracycline from Escherichia coli to Rhizobium trifolii is reported. Partially purified RP4 deoxyribonucleic acid (DNA) of the donor strain E. coli J5-3 that carried the R-factor was prepared by the lysozyme-ethylenediaminetetraacetic acid-Triton X-100 procedure and was used in transformation experiments with R. trifolii as recipient. The frequency of transformation of the R-factor into R. trifolii was 1.3 × 10⁻⁴. Dye buoyant density and sucrose gradient centrifugation of R. trifolii DNA showed that the expression of the specified drug resistance of RP4 by R. trifolii was accompanied by the acquisition of an extrachromosomal, satellite DNA component which has indistinguishable physical properties from the R-factor in the donor strain. The significance of the transformation is discussed.     

Rp4 Promotion of Transfer of a Large Agrobacterium Plasmid Which Confers Virulence

Introduction of RP4 plasmid into Agrobacterium tumefaciens promotes the transfer on solid medium of large virulence-associated plasmids from virulent donor strains to a plasmidless avirulent recipient. Exconjugants were selected for the ability to utilize octopine or nopaline as the sole source of arginine, traits which are coded for by virulence-associated plasmids in the strains employed here. All exconjugants retained the arginine auxotrophy of the recipient strain, and were resistant to ampicillin and kanamycin, drugs to which RP4 confers resistance. Five exconjugant clones from one cross were shown by alkaline sucrose gradient analysis to contain both RP4 plasmid and the large virulence-associated plasmid of the donor strain. All five exconjugants exhibited virulence on carrot, sunflower and kalanchoe plants. These results indicate that virulence and the ability to degrade octopine are plasmid-borne traits in A. tumefaciens strains 15955 and A6, and extend the evidence that large plasmids in A. tumefaciens are vectors of virulence genes.     

Isolation of nonsense suppressor mutants in Pseudomonas.

A strain of Escherichia coli harboring the drug resistance plasmid RP1 was treated with the mutagen N-methyl-N-nitro-N-nitro-N-nitrosoguanidine, and mutants were isolated in which ampicillin resistance had been lost due to an amber mutation in the plasmid. One of these mutants was again treated, and a strain was isolated in which tetracycline resistance was also lost due to an amber mutation in the plasmid. The plasmid containing amber mutations in the genes amp and tet was named pLM2. This plasmid could be transferred to strains of Pseudomonas aeruginosa, P. phaseolicola, and P. pseudoalcaligenes. Mutants resistant to ampicillin and tetracycline could not be obtained from P. phaseolicola carrying pLM2. However, strains of E. coli, P. aeruginosa, and P. pseudoalcaligenes carrying the plasmid did produce mutants simultaneously resistant to both antibiotics. All of the mutants of E. coli had developed nonsense suppressors since they became phenotypically lac+, although harboring a lac amber mutation, and formed plaques with amber mutants of phages PRR1 and PRD1 that attack organisms carrying RP1. Approximately 20% of the resistant mutants of P. aeruginosa and P. pseudoalcaligenes were sensitive to the amber mutant of PRD1. These mutants were of variable stability and grew somewhat more slowly than their parent strains. One of the suppressor mutants of P. pseudoalcaligenes, designated ERA(pLM2)S4, was used for the isolation of nonsense mutants of bacteriophage PHA6, a virus having a segmented genome of double-stranded ribonucleic acid and an envelope of lipids and proteins.     

Introduction of bacteriophage Mu into bacteria of various genera and intergeneric gene transfer by RP4::Mu.

The host range of coliphage Mu was greatly expanded to various genera of gram-negative bacteria by using the hybrid plasmic RP4::Mu cts, which is temperature sensitive and which confers resistance to ampicillin, kanamycin, and tetracycline. These drug resistance genes were transferred from Escherichia coli to members of the general Klebsiella, Enterobacter, Citrobacter, Salmonella, Proteus, Erwinia, Serratia, Alcaligenes, Agrobacterium, Rhizobium, Pseudomonas, Acetobacter, and Bacillus. Mu phage was produced by thermal induction from the lysogens of all these drug-resistant bacteria except Bacillus. Mu phage and RP4 or the RP4::Mu plasmid were used to create intergeneric recombinant strains by transfer of some genes, including the arylsulfatase gene, between Klebsiella aerogenes and E. coli. Thus, genetic analysis and intergeneric gene transfer are possible in these RP4::Mu-sensitive bacteria.     

Underexpression of Ap from R-Plasmids in Fast-Growing Rhizobium Species

The presence of the plasmid RP1 in the cells of Rhizobium leguminosarum strains Rld1, 300, and 248, R. phaseoli 1233, R. trifolii strains T1 and 6661, and R. meliloti 4013 was found to appreciably increase bacterial resistance toward kanamycin and tetracycline but not toward ampicillin. The presence of 16 other R-plasmids in R. leguminosarum was also found to either not increase or only marginally increase bacterial resistance toward ampicillin. It appears now that underexpression of the plasmid-specified ampicillin function is common to most fast- and slow-growing rhizobia.     

Intrinsic penicillin resistance in penicillinase-producing Neisseria gonorrhoeae strains

The minimal inhibitory concentrations (MICs) of ampicillin for fifty strains of beta-lactamase-producing Neisseria gonorrhoeae (PPNG) isolated in Japan ranged from 1.56 to 200 micrograms/ml, and all the strains harbored a 4.5 megadalton plasmid. These strains were classified into two groups: dicloxacillin-susceptible (28%) and -resistant group (72%). A linear correlation was found in the dicloxacillin-susceptible strains between their beta-lactamase activity and the susceptibility to ampicillin, but not in the dicloxacillin-resistant strains. This suggests that the high ampicillin resistance in PPNG is due not only to acquiring the beta-lactamase producing plasmid, but also to some intrinsic resistance of the strains. To investigate a cause of the high ampicillin resistance, the beta-lactamase-producing plasmid, pTMS1, was transferred by conjugation to a penicillin-susceptible gonococcal strain as well as to its isogenic multiply antibiotic-resistant transformants, and the susceptibility of the transconjugants to ampicillin was determined. Acquisition of pTMS1 by a penicillin-susceptible strain resulted in a 32-fold increase in resistance to ampicillin, whereas the increase was 128-fold for its isogenic strains which contain some chromosomal mutations. These results suggest that reduced permeability of the outer membrane to ampicillin underlies the high ampicillin resistance of PPNG.     

Vibrio cholerae hybrid sex factor that contains ampicillin transposon Tn1.

The ampicillin resistance transposon Tn1 was translocated from the R plasmid RP4 to the Vibrio cholerae conjugative plasmid, P. The hybrid sex factor P::Tn1 was highly transmissible and expressed the biological activities of the P factor. In addition, P::Tn1 facilitated transfer of RP4 to V. cholerae recipients. Physical studies of P::Tn1 indicated that the Tn1 transposon was added to the otherwise unaltered P plasmid.     

Lack of expression of RP4-specified β-lactamase in Azospirillum brasilense

Plasmid RP4, which normally confers resistance to ampicillin (Ap^r), tetracycline (Tc^r), and kanamycin (Km^r) to its hosts, failed to express enhanced Ap^r when transferred from Escherichia coli to Azospirillum brasilense which has its own intrinsic β-lactamase. Even in a β-lactamase-deficient mutant, A. brasilense RG-D16, no increase in β-lactamase or significant Ap^r appeared following transfer of RP4. However, A. brasilense RG (RP4) and A. brasilense RG-D16 (RP4) did exhibit Tc^r Km^r. When RP4 was transferred back from A. brasilense to E. coli all three drug resistances and β-lactamase activity were fully expressed.     

Physical size of the donor locus and transmission of Haemophilus influenzae ampicillin resistance genes by deoxyribonucleic acid-mediated transformation.

The properties of donor deoxyribonucleic acid (DNA) from three clinical isolates and its ability to mediate the transformation of competent Rd strains to ampicillin resistance were examined. A quantitative technique for determining the resistance of individual Haemophilus influenzae cells to ampicillin was developed. When this technique was used, sensitive cells failed to tolerate levels of ampicillin greater than 0.1 to 0.2 mug/ml, whereas three resistant type b beta-lactamase-producing strains could form from the colonies in 1- to 3-mug/ml levels of the antibiotic. DNA extracted from the resistant strains elicited transformation of the auxotrophic genes in a multiply auxotrophic Rd strain. For two of the donors, transformation to ampicillin resistance occurred after the uptake of a single DNA molecule approximately 104-fold less frequently than transformation of auxotrophic loci and was not observed to occur at all with the third. The frequency of transformation to ampicillin resistance was two- to fivefold higher in strain BC200 (Okinaka and Barnhart, 1974), which was cured of a defective prophage. All three clinical ampicillin-resistant strains were poor recipients, but the presence of the ampicillin resistant genes in strain BC200 did not reduce its competence. Sucrose gradients of DNA from ampicillin-resistant transformants of BC200 and from the original ampicillin-resistant strains showed that: (i) all the DNA preparations had high molecular weights; (ii) donor activity for ampicillin resistance sedimented heterogeneously and in parallel with genome DNA up to the highest molecular weights observed (100 x 106 to 200 x 106); and (iii) genetic transformation of ampicillin resistance from strain BC200-amp90383 required the physical integrity of a linearly integrated segment of DNA having a molecular weight of about 25 x 106 to 30 x 106.     

Isolation and functional-structural characteristics of Bacillus polymyxa RP4::Mucts62 transcipients]

Conjugative-like transfer of hybrid plasmid RP4::Mucts62 from Escherichia coli to plasmid-free Bacillus polymyxa was carried out. Bacillus transcipients are detected by the markers of kanamycin and tetracyclin resistance RP4 and thermal sensitivity to growth at 40-42 degrees C, determined by prophage Mucts62 in the plasmid. The technique of transception using millipore filters on solid media has been improved. For comparison with experimental samples, restriction mapping of the native plasmid in donor E. coli GA570 strain was carried out with identification of the prophage location in point 30.5 n. p. of RP4 map with counter-clockwise orientation of the right terminal towards IS21 element. Two phenotypes of bacillus transcipients selected for restriction mapping were singled out. Phenotype KmRTcRTS retains all markers of donor strain plasmid, and by the sum of restricts electrophoretically either corresponds to intact hybrid plasmid or contains deletions in RP4 sites (1-8 n. p.) adjacent to the left arm of prophage or rarely on the right arm (up to 3 n. p.). KmRTcRTS transcipients lose kanamycin resistance and on restriction map show greater prolongation of deletions from the prophage right terminal to kan RP4 gene, shortening it to 13 n. p. without involving the prophage proper. Its left terminal is retained, but 2-7 n. p. sites are deleted in the RP4 area adjacent to it. The possibility of transception in nature and horizontal routes of drug resistance dissemination among genetically remote bacteria are discussed.     

Properties of RP4, an R Factor Which Originated in Pseudomonas aeruginosa S8

RP4, an R factor derived from Pseudomonas aeruginosa S8 and specifying resistance to carbenicillin, neomycin, kanamycin, and tetracycline, could be isolated as an extrachromosomal satellite of covalently closed circular DNA (CCC-DNA) of molecular weight about 62 million and of buoyant density 1.719 g/cm(3) (60% guanine plus cytosine). When RP4 was compared by hybridization with RP1, an R factor originating in another strain of P. aeruginosa, it was shown that RP4 contains most, if not all, of the base sequences of RP1, and also additional sequences not found in RP1.     

Sequence of plasmid pBS228 and reconstruction of the IncP-1alpha phylogeny

The antibiotic resistance plasmid pBS228 has been completely sequenced, and revealed to be descended from a plasmid virtually identical to the Birmingham IncP-1alpha plasmid RK2/RP4/RP1. However, it has three additional transposon insertions, one of which is responsible for the extra antibiotic resistances conferred. Loss of kanamycin resistance, which is characteristic of most IncP-1alpha plasmids, is the result of this insertion. A second transposon causes inactivation of the mating pair formation apparatus, rendering the plasmid non-self-transmissible. Comparison with the published data for other IncP-1alpha plasmids gives insight into the recent evolutionary history of this group as well as the acquisition and transmission of one of the first ampicillin resistance transposons discovered.     

Resistance of Escherichia coli to penicillins. 8. Physiology of a class II ampicillin-resistant mutant

Class II ampicillin-resistant mutants of Escherichia coli are defined as having a twofold increase in penicillinase-mediated ampicillin resistance when determined by colony formation tests on plates. In this paper, one class II mutant has been compared to its parent strain. In liquid medium, the mutant was less resistant than the parent strain both in the absence and in the presence of R1 and R-factor mediating penicillinase activity. The penicillinase activity was found to be almost completely bound to the cells in the parent strain, whereas it was excreted to a great extent in the class II mutant strain. In liquid medium, resistance was well correlated to the cell-bound penicillinase activity, whereas the excreted penicillinases were also of great importance for survival on ampicillin plates. The mutant also had a changed resistance to a great number of other antibacterial drugs. The mutant was found to be more sensitive than the parent strain to osmotic shock, especially when treated with ethylenediaminetetraacetic acid or washed with sodium ions. However, the osmotic stability was restored by the presence of 1 mm Mg(2+) ions. The class II mutant was more sensitive than the parent strain to sodium cholate, and it adsorbed the phages T4 and T3-1 at a slower rate than did the parent strain. The two strains adsorbed T6 at the same rate. The class II phenotype could be gradually reversed by increasing concentrations of divalent cations. The pleiotropic changes in the phenotype are apparently unrelated to the specific targets for the antibacterial agents tested. They are secondary consequences of a cell envelope mutation. The findings indicate that the class II mutation mediates a structural change in the lipopolysaccharide of the cell envelope.     

IncP-1 R plasmids decrease the serum resistance and the virulence of Pseudomonas aeruginosa

Pseudomonas aeruginosa strain PAO1 was compared to PAO1 strains containing an IncP-1 R plasmid (RP1, R68, or R68.45) in an experimental mouse burn infection model. All R plasmids tested caused a 10- to 400-fold increase in mean lethal dose (LD50). The decrease in virulence produced by plasmids R68 and R68.45 was significantly greater than the decrease caused by the closely related plasmid RP1. All plasmids also led to an increased sensitivity of strain PAO1 to human serum bactericidal activity. Virulence and serum resistance of strain PAO1 were restored by curing of the entire plasmid R68.45 but not by deletions in the plasmid's transfer gene regions.     

Ampicillin Resistance in <Emphasis Type="Italic">Escherichia coli</Emphasis> by Phage Infection

BACTERIAL genes may become incorporated in the genome of phages and, as a consequence, are transmitted from one bacterium to another during lysogenization. Antibiotic resistance genes can be incorporated^2,3 and a naturally-occurring example—the phage concerned possessing genes for resistance to ampicillin—is described here. The phage lyso-genized a non-pathogenic strain of Escherichia coli (strain H) obtained from the faeces of a healthy human being who had never been treated with ampicillin. The minimum inhibitory concentration of ampicillin for this strain is 250 µg/ml.; it produced β-lactamase⁴.     

Dynamics of drug resistance of Vibrio cholerae isolated from surface water reservoirs in Siberia and the Soviet Far East in 1976-1990]

In the study on antibiotic resistance 1383 strains of El Tor Vibrio cholerae isolated from surface water reservoirs in 12 administrative territories of the Siberia and Far East within a period of 15 years were tested. The following antibiotics were used: ampicillin, streptomycin, monomycin, polymyxin, tetracycline, chloramphenicol, rifampicin and nalidixic acid. The resistance was unstable and its pattern was wave-like according to annual changes in the biological cycle. It was especially evident in regard to ampicillin, streptomycin, monomycin and polymyxin. The highest numbers of the strains were resistant to polymyxin, ampicillin and streptomycin (up to 100 per cent in some years). The lowest numbers of the strains were resistant to chloramphenicol (0.4 per cent) and tetracycline (1.9 per cent). No strains resistant to rifampicin and nalidixic acid were isolated. In some cases the antibiotic resistance level depended on the geographical zone where the strain was isolated. A direct quantitative dependence of the resistance level on the MIC was observed: the lower the MIC of the drug was, the lower the number of the strains resistant to it was. Within the 15-year period there was no general tendency to increase the resistance in V. cholerae to the antibiotics used.     

Modified RP4 and Tn5-Mob derivatives for facilitated manipulation of large plasmids in Gram-negative bacteria

We have constructed a set of RP4 (NmS/TcS) and Tn5-Mob derivatives which have applications in experiments involving mobilization of replicons in many Gram-negative organisms. The different selection markers of the RP4 and Tn5-Mob derivatives include streptomycin, chloramphenicol, gentamicin, and spectinomycin resistance as well as mercury resistance, and a constitutively expressed lacZ gene. This choice of markers allows the use of these derivatives in bacteria which are naturally resistant to many antibiotics, and in strains which contain pre-existing resistance plasmids, transposons, or antibiotic cassette insertions. In addition, a RP4 derivative carrying the sacB gene of Bacillus subtilis was constructed. This allows the selection for the loss of RP4 after it has been used to mobilize other plasmids. A Tn5-Mob-sacB derivative with a new marker (Gm) was also developed, as were vectors which take advantage of the sacB gene to facilitate replacement of existing Tn5 inserts with other Tn5 derivatives. As an example of the use of these tools, three Rhizobium leguminosarum bv. viciae VF39 plasmids which have been shown to be involved in symbiosis were differentially tagged and mobilized (individually and in various combinations) to the plasmid-free Agrobacterium tumefaciens strain UBAPF2. None of the resultant Agrobacterium strains was able to fix nitrogen in symbiosis with peas.     

Drug resistance and R plasmids in Bordetella bronchiseptica isolates from pigs

A total of 207 strains of Bordetella bronchiseptica isolated from pigs in 1978 and 1979 were tested for drug resistance and for the properties of their R plasmids. Apart from intrinsic resistance to spectinomycin, single (sulfadimethoxine), double (sulfadimethoxine and streptomycin), andt riple (sulfadimethoxine, streptomycin, and ampicillin) resistance were found in 54.1%, 1.0%, and 15.9% of the strains, respectively. All of the triple-resistance determinants were associated with mercury resistance and were conjugative. pBB1, one of these R plasmids, was identified as Fi- (F) and Spp- (no suppression of phage multiplication) type, and as a member of incompatability group IncP. The single- and double-resistance determinants were nonconjugative. pBB2, one of the double-resistance determinants, was mobilized by an R plasmid, RP4, with the high efficiency of 80% and at a frequency of 3.3% without cotransfer of RP4. The molecular weight of pBB1 and pBB2 was estimated at 36 X 10(6) and 13 X 10(6) daltons, respectively, by electron microscopy and agarose gel electrophoresis. pBB1 had five cleavage sites for EcoRI endonuclease, and four sites for HindIII. pBB2 had two EcoRI sites, one HindIII, and one BamHI site. Cells carrying pBB1 or pBB2 produced enzymic activity tha inactivated streptomycin in the presence of ATP.     

Transposition of ampicillin resistance to an enterotoxin plasmid in an Escherichia coli strain of human origin.

We examined a strain of Escherichia coli, serotype O159.H34, of human origin which produced heat-stable and heat-labile enterotoxins, was resistant to ampicillin, and produced colicin. By conjugation and transformation experiments plasmids coding for enterotoxin production (Ent), enterotoxin production and ampicillin resistance (Ap-Ent), ampicillin resistance (Ap), and colicin production were isolated. Both the Ent and Ap-Ent plasmids were autotransferring and belonged to the F-incompatibility complex. However, the Apr Ent+ transconjugants showed differences in their levels of resistance and in their abilities to propagate F-specific phages and to transfer resistance. The results suggested there was transposition from the small Ap plasmid to the Ent plasmid. The Ap-Ent plasmids were larger than the enterotoxin factor and when treated with restriction endonuclease BamHI showed an additional fragment not present in the enterotoxin plasmid. The insertion of ampicillin resistance probably occurred at different sites on the enterotoxin plasmid, resulting in the observed variation in phenotype.