Plasmids Controlling Synthesis of Hemolysin in Escherichia coli: Molecular Properties

Covalently closed extrachromosomal deoxyribonucleic acid (DNA) was isolated from alpha-hemolytic wild-type strains of Escherichia coli. Most strains examined were able to transfer the hemolytic property with varying frequencies to nonhemolytic recipient strains. Out of eight naturally isolated alphahemolytic E. coli strains, four contained a set of three different supercoiled DNAs with sedimentation coefficients of 76S (plasmid A), 63S (plasmid B), and 55S (plasmid C). The sedimentation coefficients and the contour lengths of the isolated molecules correspond to molecular weights of 65 x 10(6), 41 x 10(6), and 32 x 10(6). Three alpha-hemolytic wild-type strains carried only one plasmid with a molecular weight of 41 x 10(6), and one strain harbored two plasmids with molecular weights of 41 x 10(6) and 32 x 10(6). Alpha-hemolytic transconjugants were obtained by conjugation of E. coli K-12 with the hemolytic wild-type strains. A detailed examination revealed that plasmids with the same sizes as plasmids B and C of the wild-type strains can be transferred separately or together to the recipients. Both plasmids possess the hemolytic determinant and transfer properties. Plasmid A appears to be, at least in one wild-type strain, an additional transfer factor without a hemolytic determinant. In one case a hemolytic factor was isolated, after conjugation, that is larger in size than plasmid A and appears to be a recombinant of both plasmids B and C.     

Plasmid-determined silver resistance in Pseudomonas stutzeri isolated from a silver mine.

A silver-resistant strain of Pseudomonas stutzeri was isolated from a silver mine. It harbored three plasmids, the largest of which (pKK1; molecular weight, 49.4 X 10(6)) specified silver resistance. Plasmid pKK1 was apparently nonconjugative but could be transferred to Pseudomonas putida by mobilization with plasmid R68.45.     

Location of an ampicillin resistance transposon, Tn1701, in a group of small, nontransferring plasmids.

By restriction endonuclease cleavage mapping and electron microscopic examination of heteroduplexes, we have identified an ampicillin resistance determinant transposon, designated Tn1701, in a group of small, nontransferring plasmids which confer resistance to ampicillin (Ap), sulfonamide (Su), and streptomycin (Sm). Plasmid NTP1, which mediates Ap resistance, contains Tn1701. Recombinant plasmids NTP3 (Ap Su) and NTP4 (Ap Su Sm) contain Tn1701, indicating that they were derived by transposition of Tn1701 from NTP1 to an unrelated plasmid, NTP2 (Su Sm). The transposon Tn1701 is very similar to the known ampicillin resistance transposons Tn1, Tn2, and Tn3 in its size (3.2 x 10(6) daltons), base sequence homology observed by heteroduplex formation, restriction endonuclease cleavage sites, and possession of a short inverted repeat sequence at both ends. Like the other TnA elements, Tn1701 also specifies a type TEM beta-lactamase.     

Characterization of SmSu plasmids by restriction endonuclease cleavage and compatibility testing.

Twelve plasmids carrying genes for streptomycin and sulfonamide resistance were studied for the number and distribution of sites on the plasmid moleucles susceptible to cleavage by the restriction endonuclease EcoRI. Ten of the twelve were found to have a single cut site, one plasmid (R678) had three such sites, and plasmid PB165, which was isolated as three supercoiled deoxyribonucleic acid species with molecular weights 7.4 x 10(6), 14.7 x 10(6), and 21.4 x 10(6) was reduced to a single (linear) species of molecular weight 7.6 x 10(6) after cutting with EcoRI. We conclude that PB165 forms oligomers in Escherichia coli and that the number of copies of these per chromosome is more consistant and that the number of copies of these per chromosome is more consistent with a negative than a positive control mechanism for plasmid replication. Compatibility testing of a positive control mechanism for plasmid replication. Compatibility testing of these plasmids showed they all belong to the same incompatibility group, which we designate IncQ, suggesting that they may have come from a common ancestor.     

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.     

Plasmid complex of E. coli B-13

The plasmid complex was identified in a wild type strain B-13 of Escherichia coli. The complex was found to contain four conjugative R-plasmids / pAP24 -1 fi+, pAP24 -2 fi-, pAP24 -3 fi-, pAP24 -4 fi-/, one conjugative Col-plasmid / pAP24 -5/ and one conjugative F-like plasmid Ent/ pAP10 -2 fi+/. The molecular weight of pAP24 -1 is 53.6 X 10(6), pAP24 -2 - 40.9 X 10(6), pAP24 -3 - 73.8 X 10(6), pAP24 -4 - 51,3 X 10(6). It is suggested that an autonomous transfer factor exists in the plasmid complex.     

Comparison of plasmids in strains of Zymomonas mobilis

Four strains of Zymomonas mobilis were examined for their resistance to antimicrobial agents and found to have similar resistance profiles. Plasmid DNA was extracted and purified by CsCl dye-buoyant density centrifugation; molecular weights were determined by agarose gel electrophoresis and electron microscopy. All four strains harbored a large plasmid (46 X 10(6) Da) and a smaller plasmid (16-21 X 10(6) Da) whose molecular weight was strain dependent. Two strains, Ag11 and ATCC 10988, had smaller plasmids of unique molecular weight. Homology existed between the plasmids in the four strains as shown by cross-reaction in DNA-DNA blot hybridizations. Only one plasmid appeared unique to the host from which it was isolated.     

Translocation of an ampicillin resistance determinant within an R-factor aggregate inSalmonella panama

The molecular properties of the plasmids of a natural isolate ofSalmonella panama have been studied. This strain, Sp477, harbours 5 different plasmids: the conjugative plasmid pRI477TF (molecular weight 20 megadaltons), the two non-conjugative plasmids, pRI477A and pRI477S, coding for ampicillin and streptomycin plus sulfonamide resistance respectively (molecular weights of both 5.6 megadaltons) and two cryptic plasmids with molecular weights of 1.0 and 2.7, megadaltons respectively. After conjugal transfer toEscherichia coli the ampicillin resistance determinant was frequently found to be integrated into pRI477TF or pRI477S. The translocatable sequence on pRI477A, designated as Tn901, resembles the TnA subclass transposon TnA(1).     

Plasmids and transposable elements in Salmonella wien.

The plasmids from six clinical strains of Salmonella wien have been characterized. All the S. wien strains were found to carry three types of plasmids: an IncFI R-Tc Cm Km Ap (resistance to tetracycline, chloramphenicol, kanamycin, and ampicillin) plasmid, either conjugative or nonconjugative, of large size (90 to 100 megadaltons); an R-Ap Su Sm (resistance to ampicillin, sulfonamide, and streptomycin) plasmid of 9 megadaltons; and a very small (1.4 megadaltons) cryptic plasmid. The characteristics of conjugative R plasmids, recombinant between F'lac pro and the FI nonconjugative plasmid, indicated that regions coding for the donor phenotype were present on this plasmid. The molecular and genetic features of the R plasmids were very close to those described for the R plasmids isolated from S. wien strains of different origin. This fact supported the hypothesis of a clonal distribution of this serotype in Algeria and Europe. The analysis used to identify transposable elements showed the presence of only TnA elements, which were located on both the R-Tc Cm Km Ap and R-Ap Su Sm plasmids. They contained the structural gene for a TEM-type beta-lactamase and had translocation properties analogous to those reported for other TnA's.     

Determination of very thin pili by the bacterial drug resistance plasmid R485.

The IncX bacterial drug resistance plasmid R485 was found by electron microscopy to determine numerous very thin filaments (designated 485 pili) only 5.0 nm thick. They exhibited a characteristic helical structure (pitch, 4.6 nm), and were able to form large pseudocrystals when detached from the cell. The concomitant transfer of both pili and the sulfonamide resistance determinant of R485 to RecA strains of Escherichia coli confirmed that the pilus determinant was part of the plasmid and had not been mobilized from the chromosome of the host strain. An extensive examination failed to reveal any similar pili on strains carrying the IncX type plasmid R6K.     

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.     

Characteristics of the pKMR plasmids found in Shigella flexneri

The clinical isolate of Sh. flexneri 1b, resistant to 5 antibiotics and sulfonamides, has been studied by the method of conjugation and found to have a group of transfer-suppressed pKMR-plasmids: pKMR 204-1 (Ap Sm Tc Cm Km Su), pKMR 204-2 (Sm Km Su), pKMR 204-5 (Km Su) and pKMR 204-7 (Sm Tc Cm Km Su), whose molecular weight was 99, 71.2, 73.8 and 59.5 Md respectively. The treatment of the plasmids with restriction endonuclease BamHI has revealed that plasmids pKMR 204-2 and pKMR 204-5 are definitely related to plasmid KMR 204-1, being its deletion mutants. At the same time plasmids pKMR 204-1 and pKMR 204-7 differ in their sensitivity to endonuclease BamHI and stably coexist within the same cell, thus seeming to belong to different compatibility groups.     

Plasmids in Streptococcus lactis: evidence that lactose metabolism and proteinase activity are plasmid linked.

Populations of lactose positive (Lac+) and proteinase positive (Prt+) cells from Streptococcus lactis M18, C10, and ML3 grown at 39 degrees C gave rise to increasing proportions of Lac- Prt- clones. The deficiencies did not appear until after a number of generations at the elevated temperature, and the rate depended on the strain.Lac- Prt+ and Lac+ Prt- mutants were isolated after treatment with ethidium bromide. Plasmid deoxyribonucleic acid was isolated by cesium chloride-ethidium bromide equilibrium density gradient centrifugation from the parent cultures as well as from their Lac- Prt-, Lac- Prt+, and Lac+ Prt- mutants. Five distinct plasmid sizes of approximate molecular weights of 2,4, 8, 21, and 27 million were found in S. lactis C10, whereas the Lac- Prt- derivative lacked the 8- and 21-million-dalton plasmids, but the 8-million-dalton plasmid was present in the Lac-Att mutant. In S. lactis m18 five plasmids possessing molecular weights of about 2, 4, 10, 18 and 27 million were observed. The 10- and 18-million-dalton plasmids were not detected in the Lac- Prt- mutants, whereas the Lac- Prt+ derivative lacked only the 18-million-dalton plasmid and the Lac+ Prt- mutant lacked only the 10-million-dalton plasmid. In S. lactis ML3 five distinct plasmids, with approximate molecular weights of 2, 4, 8, 22, and 30 million, were present. The 8- and 22-million-dalton plasmids were not detected in the Lac- Prt- derivative, but the 8-million-dalton plasmid was present in the Lac- Prt+ mutant. The evidence suggests that lactose-fermenting ability and proteinase activity in these organisms are mediated through two distinct plasmids having molecular weights of 8 x 10(6) to 10 x 10(6) for proteinase activity and 18 x 10(6) to 22 x 10(6) for lactose metabolism.     

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).     

Isolation and characterization of four types of plasmids from Bacillus subtilis (natto).

Covalently closed circular deoxyribonucleic acids were found in 10 strains of Bacillus natto. The plasmids could be classified into four types on the basis of the molecular weights as well as the patterns in agarose gel electrophoresis after digestion with restriction endonucleases: (i) plasmids (seven were detected) with a molecular weight of 3.6 X 10(6); (ii) plasmids (two were detected) with a molecular weight of 4.0 X 10(6); (iii) plasmids (eight were detected) with a molecular weight of about 34 X 10(6); and (iv), a plasmid with an approximate molecular weight of 46 X 10(6). Out of the 10 plasmid-carrying strains, 6 (IFO3009, IFO3013, IFO3335, IFO13169, IAM1143, and IAM1207) harbored both type 1 and 3 plasmids; 2 (IAM1114 and IAM1168) harbored both type 2 and 3 plasmids, and IFO3936 and IAM1163 carried type 1 and 4 plasmids, respectively.     

Plasmids of Bacillus thuringiensis var. galleriae strain 612

We studied Bacillus thuringiensis var galleriae, strain 612 plasmids. B. thuringiensis cells contain double-stranded plasmid DNA molecules (ranging of about 12% from total DNA content) with buoyant density 1.59 g/cm3. Plasmid DNA content was constant during the exponential and stationary phases of bacterial growth. The plasmid fractions consist of DNA molecules with molecular weights of 5.9 x 10(6), 10.0 x 10(6), and 110.9 x 10(6) daltons (pVD1, pVD2 pVD3, respectively). Endonuclease EcoRI cuts the plasmids pVD2 and pVD3 into two and four fragments, respectivelyy, but pVDI seemed to be resistent to EcoRI treatment. We found that pVD2 and pVD3 plasmids contain a common DNA fragment with the molecular weight of 6.7 x 10(6) dalton as it was shown by restriction analysis. In contrast, the same plasmids contain the common fragment with molecular weight of 7.5 x 10(6) dalton as shown by heteroduplex analysis. Plasmid pVD3 has a transposon-like structure.     

Virulence studies based on plasmid profiles of the fish pathogen Vibrio salmonicida

Strains of Vibrio salmonicida isolated from Atlantic salmon (Salmo salar) and rainbow trout (Salmo gairdneri) suffering from cold-water vibriosis could be divided on the basis of plasmid profiles into four different categories. Of 32 strains, 19% harbored three plasmids of 24, 3.4, and 26 megadaltons (MDa), 69% harbored the 24- and 3.4-MDa plasmids but not the 2.6-MDA plasmid, and 9% harbored only the 24-MDA plasmid. The fourth category, which consisted of only one strain, harbored a plasmid of 10 MDa. In spite of different plasmid patterns, the strains of V. salmonicida were very similar with respect to biochemical reactions. The one-third of the V. salmonicida strains which were serotyped were of the same type. The 50% lethal doses, which were determined by intraperitoneal injection, ranged from 4 x 106 to 1 x 108 CFU per fish.     

Properties of R plasmid R772 and the corresponding pilus-specific phage PR772.

R plasmid R772 was isolated from a strain of Proteus mirabilis and is a self-transmissible P-1 incompatibility group plasmid having a molecular weight of about 27 x 10(6). It renders bacterial hosts resistant to kanamycin. Phage PR772 was isolated as a phage dependent on the presence of R772 in bacterial hosts. It is hexagonal-shaped with a diameter of 53 nm, has a thick inner membrane and no tail. Vaguely defined appendages are sometimes apparent at some vertices and the phage possesses double-stranded DNA. The DNA has a guanine plus cytosine molar content of 48%. The phage is sensitive to chloroform and has a buoyant density of 1.26 g cm(-3). These observations suggested that the inner membrane of the phage could contain lipid. Phage PR772 differs in morphology from the double-stranded DNA plasmid-specific phages PR4 and PRR1 which adsorb to tips and sides, respectively, of sex pili coded for by P-1 incompatibility group plasmids. Phage PR772 formed clear plaques which varied in diameter. Serologically, phages PR772 and PR4 are possibly related though very distantly, but the two phages have identical host ranges. Phage PR772 adsorbed by one of its apices to tips of sex pili coded for by plasmid R772 in Escherichia coli. It also formed plaques on Salmonella typhimurium Proteus morganii and Providence strains harbouring this plasmid as well as strains of E. coli carrying plasmids of incompatibility groups N or W. The phage produced areas of partial clearing on lawns of P. mirabilis PM5006 harbouring plasmid R772, the P-1 incompatibility group plasmid RP4, the W group plasmid RSa or the N group plasmid N3, and on lawns of Providence strain P29 carrying plasmid RP4.     

Virulence studies based on plasmid profiles of the fish pathogen Vibrio salmonicida.

Strains of Vibrio salmonicida isolated from Atlantic salmon (Salmo salar) and rainbow trout (Salmo gairdneri) suffering from cold-water vibriosis could be divided on the basis of plasmid profiles into four different categories. Of 32 strains, 19% harbored three plasmids of 24, 3.4, and 26 megadaltons (MDa), 69% harbored the 24- and 3.4-MDa plasmids but not the 2.6-MDA plasmid, and 9% harbored only the 24-MDA plasmid. The fourth category, which consisted of only one strain, harbored a plasmid of 10 MDa. In spite of different plasmid patterns, the strains of V. salmonicida were very similar with respect to biochemical reactions. The one-third of the V. salmonicida strains which were serotyped were of the same type. The 50% lethal doses, which were determined by intraperitoneal injection, ranged from 4 x 106 to 1 x 108 CFU per fish.     

Physical Properties and Mechanism of Transfer of R Factors in Escherichia coli

The physical properties of F-like and I-like R factors have been compared with those of the wild-type F factor in Escherichia coli K-12 unmated cells and after transfer to recipient cells by conjugation. The F-like R factor R538-1drd was found to have a molecular weight of 49 x 10(6), whereas the molecular weight of the I-like R factor R64drd11 was 76 x 10(6). The wild-type F factor, F1, had a molecular weight of 62 x 10(6). When conjugation experiments are performed by using donor strains carrying these derepressed F-like or I-like R factors, the transferred deoxyribonucleic acid can be isolated as a covalently closed circle from the recipient cells. This circular deoxyribonucleic acid was characterized by making use of the observation that the complementary strands of these R factors can be separated in a CsCl-poly (U, G) equilibrium gradient. The results of the strand-separation experiments show that only one of the complementary strands of the R factor is transferred from the donor to the recipient. With both the F-like and I-like R factors, this strand is the heavier strand in CsCl-poly (U, G). These results indicate that even though F-like and I-like R factors differ greatly in many properties (phage specificity, size, compatability, etc.), they are transferred by a similar mechanism.