Biomedical subjects
Publications and source records attributed to R H Olsen.
Bacteria were isolated from a landfill site previously used for disposal of chlorinated organic wastes. These soil isolates were capable of utilizing various chloroaromatic compounds. One such bacterial strain, designated Pseudomonas cepacia HCV (2,6-DCT) and growing on 2,6-dichlorotoluene, transferred this trait to a catechol-1,2-oxygenase mutant of Pseudomonas aeruginosa.
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Tn1 transposes from pRO161, a Tra- derivative of RP1, to Pseudomonas aeruginosa sex factor FP2. The acquisition of Tn1 by FP2 results in its ability to mobilize pRO161 to other bacteria. Genetic evidence presented here suggests two sequential mechanisms. Initially, transposition of Tn1 results in trans-diploidy for the Tra+ and Tra- plasmids. This subsequently allows mobilization of the Tra- R-plasmid dependent on a host recombination mechanism. Transconjugants from this mating contain either stable cointegrate R-plasmids or aggregates resulting from dissociation of the cointegrates into a Tra+ and Tra- plasmid. These aggregates have lost at least part of Tn1 from their parent FP2:Tn1 component, but now they mobilize the tra- R-plasmid from a recombination-deficient (Rec-) genetic background as well as from Rec+ donor strains. Transconjugants from these retransfer matings are aggregates. These results suggest a contribution of transposons to R-plasmid evolution and dissemination beyond the mere acquisition of resistance to a given antibiotic.
Large plasmids from Agrobacterium tumefaciens, Salmonella typhimurium, Escherichia coli, Pseudomonas putida, and Pseudomonas aeruginosa were routinely and consistently isolated using a procedure which does not require ultracentrifugation but includes steps designed to separate large-plasmid DNA from the bacterial folded chromosome. It also selectively removes fragments of broken chromosome. A variety of large plasmids was readily visualized with agarose gel electorphoresis, including five between 70 and 85 megadaltons (Mdal) in size, six between 90 and 143 Mdal, one that was larger than 200 Mdal, and one that was larger than 300 Mdal. This isolation procedure allowed initial estimation of the molecular sizes of the two IncP2 plasmids, pMG1 and pMG5, which were 312 and 280 Mdal, respectively. A standard curve for size determination by gel electrophoresis including plasmids between 23 and 143 Mdal in size did not extrapolate linearly for plasmids of the 300-Mdal size range. Unique response of different plasmids to the isolation procedure included sensitivity of IncP1 plasmids to high pH and the co-isolation of a 20-Mdal "cryptic" plasmid in conjunction.
We describe the addition to the Pseudomonas aeruginosa sex factor, FP2, of carbenicillin resistance encoded by the RP1 plasmid. This occurred in a step-wise manner as detected by variations in the characteristics of the FP2-RP1 plasmid aggregate. The addition of the carbenicillin resistance marker to FP2 facilitates estimates of FP2 transfer. Transfer frequencies for the presumed cointegrate plasmid, using carbenicillin selection, approached 10(-1) per donor bacterium. The chromosomal mobilization properties of the derived plasmid, designated pR0271, resembled those of the progenitor plasmid FP2. Plasmid pR0271 was also observed to mobilize a nontransmissible drug resistance plasmid sharing genetic homology at frequencies corresponding to those observed for chromosomal markers proximal to the origin of transfer.
We observed that Aeromonas salmonicida ARO200 will maintain either or both the Pseudomonas R-factor, pMG1, and Enterobacteriaceae R-factors. This bacterial strain, therefore, provides a unique background wherein the host ranges of Pseudomonas and Enterobacteriaceae plasmods overlap. Co-maintenance of these plasmids resulted in behavior of plasmid aggregates that allowed transfer of R-dterminants beyond the host range of the parent plasmid. We observed that the ARO200 genetic background facilitated the redistribution of B determinants among unrelated and conjugally noninterfertile gram-negative bacteria. Aberrant behavior resulting in the deletion of R-determinants for plasmids singly maintained in ARO200 was also observed. Plasmids studied included RP1, R702, IncP; Rs-a, IncW; R192.7, IncFII; R64-11, IncI; R390, IncN; and R6K, IncX.
A plasmid segregant carrying tetracycline and carbenicillin resistance markers has been isolated from R factor RP1 by transductional shortening with phage P22. The new plasmid RP1-S2, which has a molecular weight of 23 times 10-6, has lost the transfer, phage sensitivity, and neomycin resistance functions of RP1. It combines readily with a W group plasmid, R388, to form a transmissible carbenicillin and trimethoprim resistance plasmid, RWP1.
Incompatibility group P plasmids demonstrate strong entry exclusion properties. Stringent incompatibility is also observed in the absence of entry exclusion. These observations have been facilitated by the study of a nontransmissible plasmid, RP1-S2, derived from RP1 by transductional shortening. RP1-S2 retains carbenicillin and tetracycline resistances as well as loci that cause either the loss of P plasmids (incp) or a locus specifying susceptibility to curing (sinp) in the presence of a P plasmid. RP1-S2 can be mobilized by an incompatibility group W plasmid, R388, and also freely forms recombinants with R388. P, N, and W incompatibility group plasmids all encode information for the receptor of the cell wall-adsorbing phage PRD1. Based on the premise that the location of this receptor is analogous to entry exclusion factors for F-like plasmids and hence a regulated transfer region determinant, we tested fertility inhibition relationships among these plasmid groups. We detected both reciprocal and nonreciprocal fertility inhibition relationships for bacteria containing various combinations of W, N, and P group plasmids. The nonreciprocal nature of some combinations, we believe, reflects the identity of the point mutation reading to derepression of the plasmid in question. Reciprocal fertility inhibition, on the other hand, may reflect the reconstruction of a fertility inhibition system through complementation. An X incompatibility group plasmid, known to affect the fertility of an N group plasmid, was also shown to inhibit P plasmid fertility. These observations may indicate a possible evolutionary relationship(s) of plasmids unrelated by the criteria of incompatibility, pilus phage specificity, or plasmid host range.
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The Pseudomonas R factor RP1 determines an inducible tetracycline resistance similar to that described for Escherichia coli R factors. The level of RP1-determined resistance measured by minimal inhibitory concentration testing is dependent on the host bacteria and corresponds to the magnitude of decrease in tetracycline accumulation by RP1-containing organisms. The tetracycline resistance mechanism is inactivated at low temperatures. The effect of metabolic inhibitors on tetracycline accumulation by susceptible organisms under various conditions indicates a possible nonspecific effect of cellular energy metabolism on tetracycline uptake.
Several distinctive properties of PRD1, an icosahedral plasmid-dependent phage, are described. The drug-resistance plasmid-dependent host range of PRD1 extends beyond the P incompatibility group and includes gram-negative bacteria containing plasmids of incompatibility groups N and W. PRD1 phage will infect pseudomonads and Enterobacteriaceae containing either a P or W incompatibility group plasmid. PRD1 adsorbs to the cell wall of R(+) bacteria and thus its infectivity indicates cell wall alterations by these drug-resistance plasmid groups. PRD1 nucleic acid is duplex DNA with an estimated molecular weight of 24 x 10(6). The appearance of PRD1 in electron micrographs is suggestive of lipid content in addition to its buoyant density of 1.348 in CsCl and its sensitivity to chloroform. The latent period of PRD1 varies with the R(+) host bacterial strain used for growth of the phage.
R1822, a plasmid specifying multiple drug resistances, has been transferred to a variety of species representative of related and unrelated genera. The host range of the plasmid includes Enterobacteriaceae, soil saprophytes, Neisseria perflava, and photosynthetic bacteria. With the acquisition of drug resistance(s), these strains became sensitive to a small, ribonuclease-sensitive bacteriophage, designated PRR1, isolated by enrichment from sewage.
Analysis of bacteriophage CB3 infection of Pseudomonas aeruginosa strain PAT2 establishes that phage induced changes in net macromolecular synthesis are absent at nonpermissive phage growth temperatures (32 C). Alterations which are evident in the PAT2 strain at 37 C or in the fully permissive strain, PAO1C, at either warm or cold temperatures do not occur in PAT2 at low temperatures. CB3 DNA synthesis and the degradation of host DNA to approximately 78S components occur at 37 C, but are absent in PAT2 at 20 C. Nevertheless, attachment of phage DNA to host cytoplasmic material occurs under permissive and nonpermissive conditions. This binding of phage DNA at 20 C is identical in nature to phage DNA bound at 37 C. Thus, the conditional cold-sensitive PAT2 host function in the growth of CB3 is expressed subsequent to membrane binding of the infecting genomes but prior to the onset of the initiation of CB3 DNA synthesis, the inhibition of host DNA synthesis, and the transient depression in RNA synthesis which occurs in permissive cells.
Cold-sensitive restriction of Pseudomonas phage CB3 by Pseudomonas aeruginosa strain PAT2 involves some aspect of CB3 specific RNA synthesis at 20 C. Experiments using chloramphenicol treatment and RNA-DNA hybridization establish that the amount of CB3 RNA present at 20 C is consistent with the known percentage of phage yielder cells at 20 C. Thus, it appears that nonyielder cells of PAT2 synthesize little or no phage-specific mRNA. Burgess technique extracted PAT2 RNA polymerase (RNAP) is cold sensitive when assayed in vitro with CB3 DNA at 20 C. However, it is not cold sensitive when either calf thymus or PAT2 DNA are the templates for transcription. Low ionic strength assay conditions eliminate the cold sensitivity of PAT2 RNAP. The effect of low ionic environments on transcription initiation along with the in vivo and in vitro suppression of cold sensitivity by host rifampin resistance suggests that the inability of CB3 to reproduce in PAT2 at 20 C is a cold-sensitive step in host RNAP initiation. Our modified RNAP extraction procedure for PAT2 and PAO1C also results in the recovery of cold-sensitive PAT2 RNAP with respect to CB3 DNA templates and points to basic enzymological differences between the two hosts. A model is presented for the unusual influence of temperature on the initiation process of both PAT2 and PAO1C on RNAP transcription.
A new drug resistance plasmid-dependent RNA containing phage resembling coliphage f2 in its particle size and density is described. The phage, PRR1, will only productively infect some R(+) hosts containing the Pseudomonas drug resistance plasmid R1822. The membrane filter-salt elution patterns, RNase sensitivity, inactivation in low ionic strength solutions, and host range serve to distinguish PRR1 from coliphage f2 and two other Pseudomonas RNA phages, 7s and PP7.
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