Determination of the functions of hemolytic plasmid pHly152 of Escherichia coli

The alpha-hemolytic Escherichia coli strain PM152 harbors three transmissible plasmids, which have molecular weights of 65 X 10(6) (pA152), 41 X 10(6) pHly152), and 32 X 10(6) (pC152). Plasmids pHly152 and pC152 belong to incompatibility groups J2 and N, respectively. By transforming E. coli K-12 with isolated plasmids, we showed that the genetic determinant required for hemolysis was located entirely on plasmid pHly152, and a physical map of this plasmid was constructed. By transposon mutagenesis, a deoxyribonucleic acid segment of about 3.5 X 10(6) daltons was identified as being essential for hemolysis. Most of the EcoRI and HindIII fragments of the hemolytic plasmid pHly152 were cloned by using pACYC184 and RSF2124 as vectors. Two classes of Tn3-induced hemolysis-negative mutants could be complemented by recombinant plasmids carrying fragments from the hemolysis region of pHly152, whereas a third class could be restored to hemolytic activity only by recombination between the mutant plasmids and a suitable recombinant deoxyribonucleic acid. These data suggest that there are at least three clustered cistrons which are required for hemolysis. Other EcoRI and HindIII fragments of pHly152 were identified as being essential for replication, incompatibility, transfer, and restriction.     

Genetic and Biophysical Study of R Plasmids Conferring Sulfonamide Resistance in Shigella Strains Isolated in 1952 and 1956

The conjugative plasmids determining sulfonamide resistance in five Shigella strains, each isolated from a different patient, have been characterized. One S. flexneri 2a strain, isolated in 1952, harbored an fi(+) plasmid of molecular weight 53 x 10(6), which specified synthesis of F-like pili and bore determinants for sulfonamide resistance (Su) and bacteriocinogeny (Col). This plasmid was compatible with plasmids of groups F(I), F(II), I(alpha), and P. A second S. flexneri 2a strain isolated in 1952 harbored an fi(-) plasmid of molecular weight 59 x 10(6), bearing the Su determinant and compatible with all plasmids tested. This strain also harbored an fi(+) group-F(II) plasmid of molecular weight 42 x 10(6), which bore the Col determinant and specified synthesis of F-like pili. Three S. dysenteriae 2 strains isolated in 1956 carried apparently identical fi(-) plasmids of molecular weight 58 x 10(6), which bore the Su determinant, could form transconjugants in Pseudomonas but not in Proteus, and were incompatible with the P-group plasmid RP4.     

Plasmids controlling synthesis of hemolysin inEscherichia coli

Summary Plasmids of three different sizes, designated as plasmid A (mw: 65×10⁶), plasmid B (mw: 41×10⁶) and plasmid C (mw: 32×10⁶) respectively, have been isolated from various hemolytic wild-type strains ofE. coli. DNA-DNA hybridization was performed to determine their relationship. The wild-type strain, PM167a, harbours plasmids of all three sizes. Hybridization studies indicate that all three plasmids share extented sequence homologies but that plasmid A is not composed of plasmids B and C. Hybridization between plasmids of the donor strain and those of appropriate transconjugants demonstrates that in some cases plasmids with identical size are not longer completely homologous in their nucleotide sequences. This indicates that despite their defined sizes these plasmids are not stable genetic entities, but rather they undergo frequently recombination and dissociation during conjugation. In one particular transconjugant strain, K12-PM152/1, a plasmid D was found which is a stable recombined molecule of plasmids B and C of the original strain. Plasmids of size B found as the only extrachromosomal elements in a hemolytic wild-type strain (P224) and two transconjugant strains (e.g. K12-CM20 and K12-PM167/1) share extended nucleotide sequence homologies but are not identical. Little sequence homology was observed between two different hemolytic plasmids and the F and the Col Ib plasmids suggesting that the former do not belong to either the F-like or the I-like group of plasmids. Another hemolytic plasmid is F-like based on its sequence homologies with the F factor.     

Functional characteristics of plasmids of Shigella sonnei 47 strains

The phenotypic characteristics of Shigella sonnei strain 47 containing 7 plasmids of low molecular weight and 2 plasmids 60-100 Md large have been studied. The strains of Escherichia coli containing the single plasmids or plasmid groups from Shigella sonnei have been obtained by transformation and conjugation. The comparison of phenotypes of the obtained strains has helped to find the plasmid location of the determinants for streptomycin resistance (P7), genes for colicinogenicity and colicin immunity (P5), the enzymes of host cell specificity system Sso47I (P6), Sso47II (P4), and the genes for the conjugative DNA transfer (P9). Escherichia coli strains producing individual restriction enzymes SsoI and SsoII have been isolated.     

Genetic determinant of pyocin AP41 as an insert in the Pseudomonas aeruginosa chromosome.

The genetic determinant for pyocin AP41 , a bacteriocin produced by Pseudomonas aeruginosa PAF, was transferred to P. aeruginosa PAO and analyzed. By conjugation experiments, the pyocin determinant was found to be located on the chromosome, being closely linked to argG at about 45 min on the genetic map. Cloning of the pyocin AP41 gene into the plasmid R68.45 was attempted in vivo by taking advantage of its linkage at argG. R' argG+ plasmids were isolated by interspecific conjugation between P. aeruginosa and Escherichia coli recA argG strains. Some of the R' argG+ plasmids did contain the pyocin AP41 determinant. Genetic and physical analyses of these R' plasmids indicated that the pyocin AP41 determinant was located within a 2.9-kilobase extra segment found at a certain position of the chromosome of various pyocin AP41 producer strains.     

Isolation of plasmid deoxyribonucleic acid from two strains of Bacteroides.

Two clinical isolates of Bacteroides contained covalently closed circular deoxyribonucleic acid (DNA) as shown by sedimentation in an alkaline sucrose gradient, CsCl ethidium bromide equilibrium centrifugation, and electron microscopy. Bacteriodes fragilis N1175 contained a homogeneous species of plasmid DNA with a molecular weight of 25 x 10(6). Bacteroides ochraceus 2228 contained two distinct, covalently closed circular DNA elements. The larger cosedimented with the covalently closed circular DNA form of the R plasmid, R100, corresponding to a molecular weight of 70 x 10(6); the smaller sedimented as a 58S molecule with a calculated molecular weight of 25 x 10(6). The roles of these plasmids are unknown. Neither strain transferred antibiotic resistance to plasmid-negative Bacteroides or Escherichia coli, and neither produced bacteriocins active against other Bacteroides or sensitive indicator strains of E. coli.     

Comparison of the Deoxyribonucleic Acid Molecular Weights and Homologies of Plasmids Conferring Linked Resistance to Streptomycin and Sulfonamides

Bacterial strains showing linked resistance to streptomycin (Sm) and sulfonamides (Su) were chosen representing a wide taxonomic and geographical range. Their SmSu resistances were transferred to Escherichia coli K-12 and then plasmid deoxyribonucleic acid (DNA) was isolated by ethidium bromide CsCl centrifugation. The plasmid DNA was examined by electron microscopy and analyzed by sedimentation through 5 to 20% neutral sucrose gradients. Plasmid DNA from strains having transmissible SmSu resistance consisted of two or three molecular species, one of which had a molecular mass of about 5.7 Mdal (10(6) daltons), the others varying between 20 to 60 Mdal. By using transformation or F' mobilization, we isolated the SmSu-resistance determinant from any fellow resident plasmids in each strain and again isolated the plasmid DNA. Cosedimentation of each of these with a differently labeled reference plasmid DNA (R300B) showed 9 out of 12 of the plasmids to have a molecular mass not significantly different from the reference (5.7 Mdal); two others were 6.3 and 9.2 Mdal, but PB165 consisted of three plasmids of 7.4, 14.7, and 21.4 Mdal. Three separate isolations of the SmSu determinant from PB165 gave the same three plasmids, which we conclude may be monomer, dimer, and trimer, respectively. DNA-DNA hybridizations at 75 C demonstrated 80 to 93% homology between reference R300B DNA and each isolated SmSu plasmid DNA, except for the 9.2-Mdal plasmid which had 45% homology and PB165 which had 35%. All the SmSu plasmids were present as multiple copies (about 10) per chromosome. The conjugative plasmid of R300 (present as 1.3 copies per chromosome) has been shown to have negligible effect on the number of copies of its accompanying SmSu plasmid R300B. We conclude that the SmSu plasmids are closely related and probably have a common evolutionary origin.     

Conjugative plasmids in Neisseria gonorrhoeae.

A conjugation system initially discovered in beta-lactamase-producing gonococci mobilized small non-selftransmissible R plasmids encoding beta-lactamase (penicillinase) production into other gonococci, Neisseria, and Escherichia coli. This conjugation system was mediated by a separate selftransmissible plasmid of 23.9 X 10(6) daltons, pFA2. Conjugative plasmids capable of mobilizing R plasmids were also found in nearly 8% of the non-penicillinase-producing gonococci. These were similar to pFA2 in size, buoyant density, and restriction endonuclease digest patterns but were less efficient than pFA2 in mobilization of the penicillinase plasmid pFA3. The presence of conjugative plasmids in gonococci isolated before the appearance of penicillinase-producing strains indicates that a conjugation system for plasmid transfer predated the appearance of R plasmids in gonococci.     

Transposon Tn10-like tetracycline resistance determinants in Haemophilus parainfluenzae.

Tetracycline resistance (Tcr) determinants from three different strains of Haemophilus parainfluenzae expressed 10-fold higher levels of resistance when mated into Escherichia coli. No plasmid was found in any of the E. coli recipients, even in matings in which a plasmid was identified in the donor Haemophilus sp. The Tcr determinant from Haemophilus sp. caused instability of resident plasmids in the recipient E. coli: all plasmids were lost within 30 generations in antibiotic-free media. However, by serial subculture in antibiotics, stable resident plasmids were obtained which carried the Tcr determinant from Haemophilus sp. and were transferable by conjugation and transformation among E. coli strains. All Haemophilus determinants hybridized with a probe for the Tcr determinant on Tn10, which bears inducible Tcr. However, Haemophilus determinants were constitutively resistant to tetracycline in the Haemophilus donors and in the E. coli recipients. This constitutive expression was recessive to wild-type Tn10 in the same cell, indicating that the constitutive phenotype resulted from the absence of an active repressor. Restrictive enzyme analysis of various E. coli plasmid derivatives bearing a Tcr determinant from Haemophilus sp. demonstrated that the inserted DNA was of similar size (8.95 to 9.35 kilobases), close to that of Tn10. Heteroduplex analysis and DNA:DNA hybridization confirmed that the Tcr determinant from Haemophilus sp. had greater than 90% homology with the Tn10 determinant, including the DNA sequence for the repressor.     

Survey of modifying enzymes and plasmids in amikacin-resistant Serratia marcescens

Forty amikacin-resistant strains of Serratia marcescens isolated from four different hospitals (A, B, C, and D) were examined for modifying enzymes and plasmids. Twenty-one of the isolates produced acetyltransferase that modified amikacin. Eighteen of the 21 acetyltransferase-bearing isolates were from different inpatients in hospital A and the other three were from hospital C. Amikacin resistance was mediated by conjugative plasmid of 24 megadaltons in 15 of the 18 acetyltransferase-bearing isolates of hospital A and by nonconjugative plasmids, derivatives of the 24-megadalton plasmids, in the remaining three isolates of the same hospital. The 24-megadalton plasmid determined aminoglycoside acetyltransferase (6') IV. This plasmid-borne enzyme conferred amikacin resistance on S. marcescens but not on Escherichia coli K12. The frequency of transfer of the 24-megadalton plasmid from the S. marcescens isolate to E. coli K12 by conjugation was approximately 10(-7) (transconjugants/donors) and was 0.1% of that between E. coli strains. In acetyltransferase-bearing isolates from hospital C, the enzyme was mediated by a nonconjugative plasmid in one case and could not be associated with a plasmid in the remaining two cases. Neither enzymes nor plasmids could be associated with amikacin resistance of the isolates of the other two hospitals.     

IncP plasmids are most effective in mediating conjugation between Escherichia coli and streptomycetes

Mobilizable shuttle plasmids containing the origin of transfer (oriT) region of plasmid F (IncFI), ColIb-P9 (IncI1), and RP4/RP1 (IncPalpha) were constructed to test the ability of the cognate conjugation system to mediate gene transfer from Escherichia coli to Streptomyces. The conjugative system of the IncPalpha plasmids was shown to be most effective in conjugative transfer, giving peak values of (2.7 +/- 0.2) x 10(-2) S. lividans TK24 exconjugants per recipient cell. To assess whether the mating-pair formation system or the DNA-processing apparatus of the IncPalpha plasmids is crucial in conjugative transfer, an assay with an IncQ-based mobilizable plasmid (RSF1010) specifying its own DNA-processing system was developed. Only the IncPalpha plasmid mobilized the construct to S. lividans indicating that the mating-pair formation system is primarly responsible for the promiscuous transfer of the plasmids between E. coli and Streptomyces. Dynamic of conjugative transfer from E. coli to S. lividans was investigated and exconjugants starting from the first hour of mating were obtained.     

Molecular analyses of conjugative, gentamicin-resistance plasmids from staphylococcal clinical isolates

Gentamicin-resistant Staphylococcus aureus and Staphylococcus epidermidis strains which were isolated from infants with staphylococcal bacteremia were analyzed for the presence of self-transmissible gentamicin-resistance (Gmr) plasmids. Conjugative GMr plasmids of approximately 43.8-63 kilobases (kb) were found in all S. aureus strains. Inter- and intra-species transfer of Gmr plasmids by conjugation was observed from S. aureus to S. aureus and to S. epidermidis recipient strains. However, neither inter- nor intra-species transfer of gentamicin resistance by conjugation was observed with nine out of nine S. epidermidis donor strains which were mated with either S. epidermidis or S. aureus recipient strains. These conjugative Gmr plasmids were unable to comobilize a smaller (15-kb) plasmid present in all but two S. aureus clinical isolates. Many of the conjugative Gmr plasmids also carried genetic determinants for kanamycin, tobramycin, neomycin, and ethidium bromide resistance, and for beta-lactamase synthesis. EcoRI restriction endonuclease digests of the S. aureus Gmr conjugative plasmids revealed three different digestion patterns. Four EcoRI restriction endonuclease digestion fragments of 15, 11.4, 6.3, and 4.6 kb in size were common to all plasmids. These plasmids and conjugative Gmr staphylococcal plasmids from other geographical regions shared restriction digestion fragments of similar molecular weights. DNA hybridization with biotinylated S. aureus plasmid pIZ7814 DNA revealed a high degree of homology among these plasmids. A 50.9-kb plasmid from one of the nonconjugative S. epidermidis clinical isolates showed homology with the probe DNA but lacked a portion of a 6.3-kb fragment which was present in all conjugative plasmids and believed to carry much genetic information for conjugation.     

Characterization of plasmids determining hemolysin and bacteriocin production in Streptococcus faecalis 5952.

Two plasmids designated pOB1 and pOB2 were isolated from Streptococcus faecalis strain 5952 and found to have molecular weights of approximately 46 X 10(6) and 28 X 10(6), respectively. pOB1 was found to determine hemolytic activity and was transmissible, whereas pOB2 appeared to determine a bacteriocin that is specifically inhibitory to S. faecalis strains harboring the 26-megadalton plasmid pAM539.     

Plasmid R68.45 and S-a transduction by the temperate bacteriophage 59 of Erwinia carotovora 268

Temperature bacteriophage 59 of Erwinia carotovera 268 had transduced extrachromosomal DNA: plasmids of R68.45 and S-a. Before plasmid transduction experiments the suitable donor strains of indicator culture Erwinia horticola 450 harbouring R68.45 and S-a were created. The frequency of plasmid R68.45 transfer from Pseudomonas putida to E. horticola 450-8 by conjugation was equal to 5 x 10(-8) per a donor cell and in the case of S-a--from E. coli C600 for the same recipient cells--was 2 x 10(-6). Bacteriophage 59 has transduced only separate markers of plasmid R68.45, since plasmid S-a is probably transduced by the phage as an intact unit.     

Conjugal transfer and characterization of bacteriocin plasmids in group N (lactic acid) streptococci.

Thirteen bacteriocin-producing strains of group N (lactic acid) streptococci were screened for their potential to transfer this property by conjugation to Streptococcus lactis subsp. diacetylactis Bu2-60. Bacteriocin production in three strains was plasmid encoded as shown by conjugal transfer and by analysis of cured, bacteriocin-negative derivatives of the donor strains and the transconjugants. With Streptococcus cremoris strains 9B4 and 4G6 and S. lactis subsp. diacetylactis 6F7 as donors, bacteriocin-producing transconjugants were isolated with frequencies ranging from ca. 2 X 10(-2) to 2 X 10(-1) per recipient cell. Bacteriocin-producing transconjugants had acquired a 39.6-megadalton plasmid from the donor strains 9B4 and 4G6, and a 75-megadalton plasmid from the donor strain 6F7. As shown by restriction endonuclease analysis, the plasmids from strains 9B4 and 4G6 were almost identical. The plasmid from strain 6F7 yielded some additional fragments not present in the two other plasmids. In hybridization experiments any of the three plasmids strongly hybridized with each other and with some other bacteriocin but nontransmissible plasmids from other S. cremoris strains. Homology was also detected to a variety of cryptic plasmids in lactic acid streptococci.     

Incidence of plasmid DNA in Salmonella strains isolated from clinical sources in Ontario, Canada, during 1979 and 1980

Gel electrophoresis of DNA from 70 clinical strains of Salmonella revealed a heterogenous plasmid population. Plasmid DNA, ranging in molecular weight from 1.4 X 10(6) to 145 X 10(6), was demonstrated in 26 of 32 antibiotic-resistant strains. Several resistant strains carried up to six plasmids; however, of these, five strains which were multiply resistant contained a single plasmid of molecular weight 54 X 10(6) to 145 X 10(6). Only one incompatibility group H2 (IncH2) plasmid (pDT28) was detected in a strain of S. heidelberg; thus, this represents a reduction in the prevalence of these plasmids in Ontario Salmonella strains since 1974. The pDT28 plasmid resembled other IncH2 plasmids by its high molecular weight (145 X 10(6) ) and by virtue of its temperature-sensitive mode of transfer, resistance to tellurium, and inhibition of coliphage development. Of the 38 antibiotic-susceptible Salmonella strains, approximately half contained plasmids, ranging in molecular weight from 1.4 X 10(6) to 60 X 10(6). The plasmid-containing antibiotic-susceptible strains carried either a group of two to four small plasmids, with molecular weights less than 4.5 X 10(6), or a single large plasmid of molecular weight 23 X 10(6) or 60 X 10(6).     

Transfer of multiple drug resistance plasmids between bacteria of diverse origins in natural microenvironments

Plasmids harboring multiple antimicrobial-resistance determinants (R plasmids) were transferred in simulated natural microenvironments from various bacterial pathogens of human, animal, or fish origin to susceptible strains isolated from a different ecological niche. R plasmids in a strain of the human pathogen Vibrio cholerae O1 E1 Tor and a bovine Escherichia coli strain were conjugated to a susceptible strain of the fish pathogenic bacterium Aeromonas salmonicida subsp. salmonicida in marine water. Conjugations of R plasmids between a resistant bovine pathogenic E. coli strain and a susceptible E. coli strain of human origin were performed on a hand towel contaminated with milk from a cow with mastitis. A similar conjugation event between a resistant porcine pathogenic E. coli strain of human origin was studied in minced meat on a cutting board. Conjugation of R plasmids between a resistant strain of the fish pathogenic bacterium A. salmonicida subsp. salmonicida and a susceptible E. coli strain of human origin was performed in raw salmon on a cutting board. R plasmids in a strain of A. salmonicida subsp. salmonicida and a human pathogenic E. coli strain were conjugated to a susceptible porcine E. coli strain in porcine feces. Transfer of the different R plasmids was confirmed by plasmid profile analyses and determination of the resistance pattern of the transconjugants. The different R plasmids were transferred equally well under simulated natural conditions and under controlled laboratory conditions, with median conjugation frequencies ranging from 3 x 10(-6) to 8 x 10(-3). The present study demonstrates that conjugation and transfer of R plasmids is a phenomenon that belongs to the environment and can occur between bacterial strains of human, animal, and fish origins that are unrelated either evolutionarily or ecologically even in the absence of antibiotics. Consequently, the contamination of the environment with bacterial pathogens resistant to antimicrobial agents is a real threat not only as a source of disease but also as a source from which R plasmids can easily spread to other pathogens of diverse origins.     

Transfer of multiple drug resistance plasmids between bacteria of diverse origins in natural microenvironments.

Plasmids harboring multiple antimicrobial-resistance determinants (R plasmids) were transferred in simulated natural microenvironments from various bacterial pathogens of human, animal, or fish origin to susceptible strains isolated from a different ecological niche. R plasmids in a strain of the human pathogen Vibrio cholerae O1 E1 Tor and a bovine Escherichia coli strain were conjugated to a susceptible strain of the fish pathogenic bacterium Aeromonas salmonicida subsp. salmonicida in marine water. Conjugations of R plasmids between a resistant bovine pathogenic E. coli strain and a susceptible E. coli strain of human origin were performed on a hand towel contaminated with milk from a cow with mastitis. A similar conjugation event between a resistant porcine pathogenic E. coli strain of human origin was studied in minced meat on a cutting board. Conjugation of R plasmids between a resistant strain of the fish pathogenic bacterium A. salmonicida subsp. salmonicida and a susceptible E. coli strain of human origin was performed in raw salmon on a cutting board. R plasmids in a strain of A. salmonicida subsp. salmonicida and a human pathogenic E. coli strain were conjugated to a susceptible porcine E. coli strain in porcine feces. Transfer of the different R plasmids was confirmed by plasmid profile analyses and determination of the resistance pattern of the transconjugants. The different R plasmids were transferred equally well under simulated natural conditions and under controlled laboratory conditions, with median conjugation frequencies ranging from 3 x 10(-6) to 8 x 10(-3). The present study demonstrates that conjugation and transfer of R plasmids is a phenomenon that belongs to the environment and can occur between bacterial strains of human, animal, and fish origins that are unrelated either evolutionarily or ecologically even in the absence of antibiotics. Consequently, the contamination of the environment with bacterial pathogens resistant to antimicrobial agents is a real threat not only as a source of disease but also as a source from which R plasmids can easily spread to other pathogens of diverse origins.     

Two conjugation systems associated with Streptococcus faecalis plasmid pCF10: identification of a conjugative transposon that transfers between S. faecalis and Bacillus subtilis.

The tetracycline resistance plasmid pCF10 (58 kilobases [kb]) of Streptococcus faecalis possesses two separate conjugation systems. A 25-kb region of the plasmid (designated TRA) was shown previously to determine pheromone response and conjugation functions required for transfer of pCF10 between S. faecalis cells (P. J. Christie and G. M. Dunny, Plasmid 15:230-241, 1986). When S. faecalis cells were mixed with Bacillus subtilis in broth, tetracycline resistance was transferred from S. faecalis. The tetracycline-resistant B. subtilis cells contained a 16-kb region of pCF10 (distinct from TRA) that carried the tetracycline resistance determinant (Tetr). This Tetr element was found to transfer between S. faecalis and B. subtilis strains in the absence of plasmids. Genetic and molecular techniques were used to establish locations of the element at several different sites on the B. subtilis chromosome. The Tetr element could be transferred in filter matings from B. subtilis to S. faecalis strains and between recombination-proficient and -deficient S. faecalis strains in the absence of any plasmid DNA. The transfer required direct cell-to-cell contact and was not inhibited by DNase. The Tetr element was shown to transpose from the S. faecalis chromosome to various locations within the hemolysin plasmid pAD1. Together, the data indicate that the Tetr element, termed transposon Tn925, is very similar to the conjugative transposon Tn916 in both structure and function. A derivative of Tn925, containing transposon Tn917 inserted into a site approximately 3 kb from one end, exhibited elevated transfer frequencies and may provide a useful means for delivering Tn917 by conjugation into various gram-positive species.     

Molecular and genetic studies of an R factor system consisting of independent transfer and drug resistance plasmids

Certain genetic, structural, and biochemical properties of a class 2 R-factor system consisting of the conjugally proficient transfer plasmid I and the naturally occurring non-conjugative tetracycline (Tc) resistance plasmid 219 are reported. I and 219 exist as separate plasmid deoxyribonucleic acid (DNA) species in both Escherichia coli and Salmonella panama, having molecular weights of 42 x 10(6) and 5.8 x 10(6), respectively. The buoyant densities of I and 219 are 1.702 and 1.710 g/cm(3), respectively, in neutral cesium chloride. Although the Tc resistance plasmid is not transmissible in a normal conjugal mating, it is mobilized in a three-component mating by plasmid I and by certain other conjugative plasmids of the fi(+) or fi(-) phenotype. Mobilization does not appear to involve intermolecular recombination between plasmids, and no covalent linkage of resistance markers and fertility functions is observed. Transformation of CaCl(2)-treated E. coli by plasmid DNA is shown to be a useful procedure for studying the biological properties of different plasmid molecular species that have been fractionated in vitro, and for selectively inserting non-self-transmissible plasmids into specific bacterial strains. The effects of tetracycline on the rate of protein synthesis carried out by plasmid 219 were studied by using isolated E. coli minicells into which this plasmid had segregated. Consistent with the results of earlier investigations showing the inducibility of plasmid-mediated Tc resistance in E. coli, the antibiotic was observed to stimulate protein synthesis in minicells carrying the plasmid 219 and totally inhibit (3)H-leucine incorporation by minicells lacking the Tc resistance marker. Five discrete polypeptide species were synthesized by minicells carrying plasmid 219; exposure of minicells or parent bacteria to Tc resulted in specific and reproducible changes in polypeptide synthesis patterns.