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.     

Isolation and characterization of four plasmids from Bacillus subtilis.

Nineteen Bacillus subtilis isolates obtained from type culture collections were examined for the presence of covalently closed circular duplex deoxyribonucleic acid molecules by the technique of cesium chloride-ethidium bromide density gradient centrifugation. Four of the 19 strains tested carried covalently closed circular molecules. Two of these strains (IFO3022, IFO3215) harbored a similar plasmid with a molecular weight of 5.4 X 10(6). The other two strains (IAM1232, IAM1261) carried 4.9 C 10(6)-and 5.3 X 10(6)-dalton plasmids, respectively. These plasmid-harboring strains did not show phenotypic traits such as antibiotic resistance orbacteriocin production. The plasmid deoxyribonucleic acids were digested by three restriction endonucleases, EcoRI, HindIII, and BamNI, and were classified into three different types from their electrophoretic patterns in agarose gels.     

Plasmids, loss of lactose metabolism, and appearance of partial and full lactose-fermenting revertants in Streptococcus cremoris B1.

The unstable ability to metabolize lactose (lac) via the phosphoenolpyruvate-phosphotransferase system (PTS) was examined in Streptococcus cremoris B1. The presence of functional lactose-specific PTS enzymes was correlated with the presence of a distinct plasmid species. Characterization of deoxyribonucleic acid extracted from lactose-positive (Lac+) S. cremoris B1 revealed two plasmids having molecular weights of 9 X 10(6) and 36 X 10(6). An acriflavine (BC1)-induced, lactose-negative (Lac-) mutant possessed no plasmids and was devoid of all three lac-specific PTS enzymes. A Lac- mutant (DA2) isolated by growing at elevated temperatures only possessed the 9 X 10(6)-dalton plasmid and also lacked the lac PTS enzymes. A spontaneous Lac- mutant possessed both the 9 X 10(6)-and 36 X 10(6)-dalton plasmids. This mutant displayed FIII-lac and phospho-beta-D-galactosidase (P-beta-gal) activity but was deficient in EII-lac activity. The spontaneous Lac- strain reverted to both full and partial lactose-fermenting phenotypes having FIII-lac, EII-lac, and P-beta-gal activities. BC1 and DA2 Lac- mutants reverted only to the partial lactose-fermenting phenotype having P-beta-gal activity; EII-lac and FIII-lac activities were absent. The results indicate that the genetic determinants for EII-lac, FIII-lac, and P-beta-gal are located on the 36 X 10(6)-dalton plasmid in S. cremoris B1. Evidence for a second chromosomally associated P-beta-gal gene operating in the partial lactose-fermenting revertants is also presented.     

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.     

Stability in Escherichia coli of an antibiotic resistance plasmid from Bacteroides fragilis.

A Bacteroides fragilis strain resistant to penicillin G, tetracycline, and clindamycin was screened for the presence of plasmid deoxyribonucleic acid (DNA). Agarose gel electrophoresis of ethanol-precipitated DNA from cleared lysates of this strain revealed two plasmid DNA bands. The molecular weights of the plasmids were estimated by their relative mobility in agarose gel and compared with standard plasmids with known molecular weights. The molecular weights were 3.40 +/- 0.20 x 10(6) and 1.95 +/- 0.05 x 10(6) for plasmids pBY1 and pBY2, respectively. Plasmid DNA purified by cesium chloride-ethidium bromide gradient centrifugation was used to transform a restriction- and modification-negative strain of Escherichia coli. Penicillin G- and tetracycline-resistant transformants were screened for the presence of plasmid DNA. A plasmid band corresponding to a molecular weight of 1.95 x 10(6) was present in all transformants tested. Curing experiments demonstrated that the plasmid, referred to as pBY22 when present in transformants, was responsible for penicillin G and tetracycline resistance. Plasmid pBY22 was mobilized and transferred to other E. coli strains by plasmid R1drd-19. Stability of pBY22 was examined in different E. coli strains and was shown to be stably maintained in both restriction-negative and restriction-positive strains. Unexpectedly, pBY2 and pBY22 were resistant to digestion by 12 different restriction endonucleases.     

Isolation and partial characterization of four plasmids from antibiotic-resistant thermophilic bacilli.

Twenty-nine antibiotic-resistant isolates of thermophilic bacilli were examined for the presence of covalently closed circular duplex DNA molecules by agarose-gel electrophoresis and caesium chloride-ethidium bromide density gradient centrifugation. Five of the 29 strains tested contained covalently closed circular molecules. Two of the streptomycin-resistant strains contained the same two plasmids: pAB118A of molecular weight 4.9 X 10(6) (7.0 kilobases) and pAB118B of molecular weight 3.0 X 10(6) (4.3 kilobases). Two of the tetracycline-resistant strains each contained a plasmid (pAB124) of molecular weight 2.9 X 10(6) (4.14 kilobases), while a third harboured a small plasmid (pAB128) of molecular weight 2.5 X 10(6) (3.57 kilobases). These plasmids were digested with 19 different restriction endonucleases and the numbers of cleavage sites were determined. Transformation of Bacillus subtilis (168 (Trp-) with purified plasmid DNA indicated that pAB124 conferred tetracycline resistance on the host.     

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.     

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.     

Isolation of extrachromosomal deoxyribonucleic acids from extremely thermophilic bacteria

Eight strains of thermophilic bacteria were examined for the presence of covalently closed circular deoxyribonucleic acid molecules by caesium chloride-ethidium bromide density gradient centrifugation. Four of the eight strains tested, Thermus flavus BS1, AT61, AT62 and Thermus thermophilus HB8 carried covalently closed circular DNA molecules. Thermus flavus BS1 haboured two species of plasmids with molecular weights of 6.1 X 10(6) and 17.0 X 10(6) as determined by electron microscopy. Thermus thermophilus HB8, T. flavus AT61 and T. flavus AT62 carried plasmids with molecular weights of 6.2 X 10(6), 6.6 X 10(6) and 6.6 X 10(6), respectively. Plasmids from T. flavus AT61 and AT62 were indistinguishable in their electrophoretic patterns in agarose or acrylamide gel after digestion with various restriction endonucleases. This is the first evidence for the presence of plasmids in extremely thermophilic bacteria, though their functions are unknown.     

Circular deoxyribonucleic acid from Shigella dysenteriae Y6R

Circular deoxyribonucleic acid was isolated from Shigella dysenteriae Y6R and was found to consist of six species having molecular weights of 10(6), 1.3 x 10(6), 2.6 x 10(6), 3.8 x 10(6), 20 x 10(6), and 24 x 10(6) daltons. These size classes were partially resolved by sucrose density gradient centrifugation. The minicircles (10(6) and 1.3 x 10(6)) were found to have a buoyant density in CsCl of 1.710 g/ml. The 3.8 x 10(6) dalton class had a density of 1.707 g/ml. The two largest species had a density of 1.702 g/ml. Two other strains, S. sonnei II and S. dysenteriae 60, also contained circular deoxyribonucleic acid.     

Genetic loci of hemolysin production in Streptococcus faecalis subsp. zymogenes.

Two plasmids corresponding to molecular weights of 38.5 X 10(6) and 3.6 X 10(6) have been identified in Streptococcus faecalis subsp. zymogenes strain X-14. The larger plasmid is required for hemolysin-bacteriolysin production. Strain L2, a nonlytic nitrosoquanidine mutant of strain X-14, still harbors the hemolytic plasmid and produces the lysin component, but not the activator component, of the lytic system. Conjugal transfer of this plasmid from strain L2 to plasmid-free strains and strains cured of the 38.5-megadalton plasmid gives rise to hemolytic recipients. This implicates a gene in hemolysin production at a site other than the 38.5-megadalton plasmid.     

Extrachromosomal Elements in Group N Streptococci

The deoxyribonucleic acid (DNA) of Streptococcus lactis C2, S. cremoris B(1), and S. diacetilactis 18-16 was labeled by growing cells in Trypticase soy broth containing (3)H-labeled thymine. The cells were gently lysed with lysozyme, ethylenediaminetetraacetic acid, and sodium lauryl sulfate. The chromosomal DNA was separated from plasmid DNA by precipitation with 1.0 M sodium chloride. The existence of covalently closed circular DNA in the three organisms was shown by cesium chloride-ethidium bromide equilibrium density gradient centrifugation of the cleared lysate material. In an attempt to correlate the loss of lactose metabolism with the loss of plasmid DNA, lactose-negative mutants of these organisms were examined for the presence of extrachromosomal particles. Covalently closed circular DNA was detected in the lactose-negative mutants of S. lactis C2 and S. diacetilactis 18-16. In S. cremoris B(1), however, no covalently closed circular DNA was observed by using cesium chloride-ethidium bromide gradients. Electron micrographs of the satellite band material from S. lactis C2 and its lactose-negative mutant confirmed the presence of plasmid DNA. Three distinct plasmids having approximate molecular weights of 1.3 x 10(6), 2.1 x 10(6), and 5.1 x 10(6) were observed in both organisms.     

Generation of miniplasmids from copy number mutants of the R plasmid NR1.

Small, closed circular deoxyribonucleic acid molecules, called miniplasmids, were observed in Escherichia coli harboring copy number mutants of the R plasmid NR1 after growth in medium containing tetracycline. The level of tetracycline resistance conferred by the copy mutant plasmids was lower (3 to 6 microgram/ml) than that conferred by NR1 (100 MICROGRAM/ML). The presence of the miniplasmid enhanced the level of tetracycline resistance conferred by the copy mutant. Miniplasmids of molecular weights 4 X 10(6) to 13 X 10(6) were found. They carried no antibiotic resistance markers and could be eliminated by growth in the presence of chloramphenicol and/or streptomycin-spectinomycin. Studies with the restriction endonucleases EcoRI and Sal I indicated that the miniplasmids are derived from the region of the copy mutant plasmids that contains the origin for replication of the resistance transfer factor. There were approximately 12 copies of the miniplasmid per chromosome, compared with 3 and 6 copies of the copy mutants of NR1. The miniplasmids appeared to be incompatible with the copy mutant plasmids.     

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.     

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.     

Bacillus pumilus plasmid pPL10: properties and insertion into Bacillus subtilis 168 by transformation.

Bacillus pumilus ATCC 12140 harbors 10 or more copies per chromosome of each two small plasmids. Variants of this strain were isolated which were sensitive to a killing activity produced by the plasmid-containing parent. Each of 24 such sensitive (S) variants tested lacked detectable levels of supercoiled deoxyribonucleic acid. Transduction of S variants to the Kill+ phenotype was performed using phage PBP1 propagated on a mutant of ATCC 12140, designated strain L10, that remained Kill+ but retained only a single plasmid species (plasmid pPL10; molecular weight, approximately 4.4 X 10(6), approximately 20 copies per chromosome; RHO = 1.698). Resulting Kill+ transductants of S variants contained a single plasmid species having a size and copy number comparable to that of pPL10. Transfer of pPL10 from strain L10 TO B. pumilus strain NRS 576 was accomplished by transduction with selection for the Kill+ phenotype. Strain NRS 576 naturally harbors about two copies per chromosome of a 28-million-dalton plasmid, pPL576. In Kill + transductants of NRS 576, plasmids pPL10 and pPL576 stably coexisted at a ratio of about 11 molecules of pPL10 to 1 molecule of pPL576. Therefore, pPL576 and pPL10 are compatible plasmids. B. subtilis 168 is naturally resistant to pPl10- determined killing activity. Plasmid pPl10 was therefore inserted into B-subtilis 168 by transformation, using an indirect selection procedure and a spoB mutant as recipient. The plasmid is stably maintained at an estimated 10 copies per chromosome in the spore- recipient and in spore+ transformants. pPL10 is sensitive to cleavage by the endonucleases Hind III and EcoR1.     

Isolation and characterization of linear deoxyribonucleic acid plasmids from Kluyveromyces lactis and the plasmid-associated killer character.

Two linear deoxyribonucleic acid plasmids, designated pGK11 and pGK12, were isolated from the yeast Kluyveromyces lactis IFO 1267. pGK11 and pGK12 had molecular weights of 5.4 X 10(6) and 8.4 X 10(6), respectively. Both plasmids possessed the same density of 1.687 g/cm3, lighter than the densities of mitochondrial (1.692 g/cm3) and nuclear (1.699 g/cm3) deoxyribonucleic acids. A restriction map of pGK11 was constructed from digestions by EcoRI, HindIII, PstI, and BamHI. pGK12 was cleaved by EcoRI into seven fragments and by BamHI into two fragments K. lactis IFO 1267 killed Saccharomyces cerevisiae sensitive and killer strains and certain strains of Saccharomyces italicus, K. lactis, Kluyveromyces thermotolerans, and K. vanudenii. All K. lactis strains lacking the pGK1 plasmids were nonkillers. A hybrid was constructed between K. lactis IFO 1267 and a nonkiller K. lactis strain lacking the plasmids and subjected to tetrad analysis after sporulation. The killer character was extrachromosomally transmitted in all tetrads in association with the pGK1 plasmids. The double-stranded ribonucleic acid killer plasmid could not be detected in any K. lactis killer strains. It is thus highly probable that the killer character is mediated by the linear deoxyribonucleic acid plasmids. A single chromosomal gene was found which was responsible for the resistance to the K. lactis killer.     

Plasmid deoxyribonucleic acid and translucent-to-opaque variation in Mycobacterium intracellulare 103.

After treatment with mitomycin D and other antibacterial agents, a translucent, smooth-colony-forming mycobacterium, isolated from sputum and designated as Mycobacterium intracellulare strain 103, gave rise to variants forming opaque colonies. These opaque variants were more sensitive streptomycin, kanamycin, viomycin, and rifampin than were the wild-type translucent variants. Plasmid deoxyribonucleic acids taken from translucent strain cells and from cells of certain opaque variants were analyzed by agarose gel electrophoresis. Two plasmids of molecular weights of approximately 2 x 10(6) and 50 x 10(6), respectively, were found in the wild-type translucent cells; one of them, the 2 x 10(6)-molecular-weight plasmid, was always missing from deoxyribonucleic acids of the opaque variant cells. The results suggested that translucent colonial appearance and antibiotic resistance of the strain are plasmid-determined functions.     

Isolation of plasmid deoxyribonucleic acid from Pseudomonas putida.

Conditions suitable for reproducible recovery of covalently closed circular deoxyribonucleic acid from strains of Pseudomonas putida containing degradative plasmids (CAM, SAL, OCT, etc.) have been defined. These degradative plasmids could not be isolated by the usual procedure, whereas RP1, an R factor of the P group, present in the isogenic strain of P. putida, was isolated equally well by either the usual procedure or the modified procedure. Characterization by electron microscopy of RP1 deoxyribonucleic acid confirmed the molecular weight (about 40 X 10(6)) previously determined by sucrose gradient centrifugation.     

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.