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.     

Temperate Bacteriophage Infectious for Asporogenic Variants of Bacillus pumilus

Bacillus pumilus strain NRRL B-3275 is lysogenic for an inducible, nondefective temperate bacteriophage phi75. phi75 infects and lysogenizes several asporogenic mutants of B. pumilus strain NRS 576 but does not productively infect the spore(+) parent. phi75 DNA is a linear duplex with a mol wt of about 29 x 10(6) and a buoyant density of 1.701 g/cm(3). The location of the phi75 prophage attachment site on the chromosome of both host strains is adjacent to a lysine marker. The apparent order is phi75 att lys trp.     

Some Properties of the PBP1 Transduction System in Bacillus pumilus

Bacteriophage PBP1 is a flagella-specific virus that performs generalized transduction in strains of Bacillus pumilus. PBP1 is morphologically and serologically distinct from two other flagella-specific phages, PBS1 and SP-15, which perform generalized transduction in certain Bacillus species. The DNA extracted from PBP1 particles has a buoyant density of 1.690 g/cm(3) in cesium chloride gradients, a melting temperature of 86.1 C, and a sedimentation velocity of 47S in neutral sucrose gradients. Assuming the molecule is a linear duplex, PBP1 DNA has a molecular weight of approximately 76 x 10(6). In two strains of B. pumilus which are sensitive to both PBP1 and PBS1, co-transducible genetic markers are more tightly linked by PBS1 transduction than by PBP1 transduction. The size of the fragment of bacterial DNA carried by PBP1-transducing particles, inferred from transduction studies and sedimentation analysis of viral DNA, suggests that PBP1 may be useful for genetic studies of extrachromosomal DNA elements present in two strains of B. pumilus. Genetic exchange of chromosomally located genes between the plasmid(+) and plasmid(-)B. pumilus strains NRS 576 and NRRL B-3275 has been demonstrated by PBP1 transduction.     

Low-frequency, pbsi-mediated plasmid transduction in Bacillus pumilus.

Three bacteriophages were tested for ability to transduce the plasmid of pPL10 between W mutant derivatives of Bacillus pumilus NRS 576. Phage PBP1- and PMB1-generated plasmid transductants occurred at about 10% the frequency of transductants for a chromosome marker. Phage PBS1-generated plasmid transductants occurred at less than 0.1% the frequency of transductants for a chromosome marker. Possible reasons for the extremely reduced capacity of PBS1 to generate plasmid transductants are discussed.     

Evidence for a nonrandom base sequence in a Bacillus pumilus plasmid: EcoR1 endonuclease digestion of pPL576.

EcoR1 endonuclease digested the Bacillus pumilus plasmid pPL576 (molecular weight similar to 28 X 10-6) into three distinct size classes of linear fragments. The molecular weights of the fragments are 13.0 X 10-6, 0.5 X 10-6, and 6.5 X 10-6 by sucrose gradient analysis. By electron microscope analysis the three fragments account for about 99% of the intact plasmid. These results indicate that pPL576 molecules contain a nonrandom base sequence, and are consistent with the interpretation that pPL576 is autonomous and not the result of cyclization of random chromosome fragments.     

Detection of nonintegrated plasmid deoxyribonucleic acid in the folded chromosome of Escherichia coli: physiochemical approach to studying the unit of segregation.

Physiocochemical evidence presented indicates plasmid deoxyribonucleic acid (DNA) can associate with host chromosome without linear insertion of the former into the latter. This conclusion is based on the observation that covalently closed circular (CCC) plasmid DNA can cosediment with undegraded host chromosome in a neutral sucrose gradient. When F plus bacteria are lysed under conditions that preserve chromosome, approximately 90% of CCC F sex factor plasmid (about 1% of the total DNA) is found in folded chromosomes sedimenting at rates between 1,500 and 4,000s. The remaining 10% of the CCC F DNA sediments at the rate (80S) indicative of the free CCC plasmid form. Reconstruction experiments in which 80S, CCC F DNA is added to F plus or F minus bacteria before cell lysis show that exogenous F DNA does not associate with folded chromosomes. In F plus bacteria, F plasmid is harbored at a level of one or two copies per chromosomal equivalent. In bacteria producing colicin E1, the genetic determinant of this colicin, the Col E1 plasmid, is harbored at levels of 10 to 13 copies per chromosomal equivalent; yet, greater than 90% of these plasmids do not cosediment with the 1,800S species of folded chromosome. However, preliminary evidence suggests one or two Col E1 plasmids may associate with the 1,800S folded chromosome. Based on evidence presented in this and other papers, we postulate F plasmid can link to folded chromosome because the physicochemical structure of the plasmid resembles a supercoiled region of the chromosome and, therefore, is able to interact with the ribonucleic acid that stabilizes the folded chromosome structure. Implications of this model for F plasmid replication and segregation are discussed.     

Plasmid in Bacillus pumilus and the Enhanced Sporulation of Plasmid-Negative Variants

Approximately 3% of the deoxyribonucleic acid (DNA) of Bacillus pumilus NRS576 can be isolated as covalently closed, circular duplex molecules of homogeneous size. The mol wt of the 576 plasmid is approximately 30 million. NRS576 (plasmid(+)) is oligosporogenic; less than 1% of the cells form spores during incubation in liquid AK sporulation medium. Variants that form spores at a high frequency (greater than 50% spores in 24 h) occur spontaneously at a frequency of approximately 10(-5). More than 25 such variants have been examined and all lack detectable plasmid DNA. A relationship appears to exist between the oligosporogenic properties of NRS576 and the presence of the 576 plasmid.     

Recombination between compatible plasmids containing homologous segments requires the Bacillus subtilis recE gene product.

Plasmid pSL103 was previously constructed by cloning a Trp fragment (approximately 2.3 X 10(6) daltons) from restriction endonuclease EcoRI-digested chromosome DNA of Bacillus pumilus using the neomycin-resistance plasmid pUB110 (approximately 2.8 X 10(6) daltons) as vector and B. subtilis as transformation recipient. In the present study the EcoRI Trp fragment from pSL103 was transferred in vitro to EcoRI fragments of the Bacillus plasmid pPL576 to determine the ability of the plasmid fragments to replicate in B. subtilis. Endonuclease EcoRI digestion of pPL576 (approximately 28 X 10(6) daltons) generated three fragments having molecular weights of about 13 X 13(6) (the A fragment), 9.5 X 10(6) (B fragment, and 6.5 X 10(6) (C fragment). Trp derivatives of pPL576 fragments capable of autonomous replication in B. subtilis contained the B fragment (e.g., pSL107) or both the B and C fragments (e.g., pSL108). Accordingly, the B fragment of pPL576 contains information essential for autonomous replication. pSL107 and pSL108 are compatible with pUB110. Constructed derivatives of the compatible plasmids pPL576 and pUB110, harboring genetically distinguishable EcoRI-generated Trp fragments cloned from the DNA of a B. pumilus strain, exhibited relatively high frequency recombination for a trpC marker when the plasmid pairs were present in a recombination-proficient strain of B. subtilis. No recombination was detected when the host carried the chromosome mutation recE4. Therefore, the recE4 mutation suppresses recombination between compatible plasmids that contain homologous segments.     

Isolation and characterization of Escherichia coli chromosomal mutants affecting plasmid copy number.

We have isolated chromosomal mutants of an Escherchia coli K-12 strain that maintain higher levels of an F' plasmid. The mutants are designated as plasmid copy number (pcn) mutants. They were detected by selecting for increased lactose fermentation in bacteria deleted for the lac operon but harboring an F'lacI,P pro+ plasmid. When examined for the amount of F' plasmid deoxyribonucleic acid (DNA) by the dye-CsCl isopycnic technique, the mutants show two to seven times as much covalently closed, circular (CCC) DNA as does the parental strain. The increased plasmid level in one mutant strain (pcn-24) was confirmed by DNA-DNA hybridization; however, this latter technique indicated about a twofold lower increase when compared with the increase measured for pcn-24 by the dye-CsCl technique. In mutant pcn-24 the increased amount of F' DNA reflects a proportional increase in monomeric-size plasmid molecules because oligomeric forms are not found. Also, in mutant pcn-24 the extra CCC plasmid copies do not seem to be randomly distributed throughout the cell's cytoplasm but appear complexed in situ with their host's folded chromosome. In all pcn mutants examined to date, the classical sex factor F is maintained at normal levels, whereas the viral plasmid Pl CM is maintained at two to three times the normal level. In all 17 pcn mutants isolated, the pcn mutation maps on the chromosome and not on the plasmid. Finally, the absolute amount of CCC F' DNA detectable in lysates of the six different pcn mutants examined decreased 50 to 90% upon incubation of the lysate at 37 C. In contrast, no loss of CCC DNA occurs when lysates of the parental F' strain are incubated at 37 C.     

Physical properties of plasmid Mor174, which determines bacteriocin production in Proteus morganii 174.

Plasmid Mor174 has a molecular weight of 3.6 X 10(6) and a buoyant density of 1.6994 g/cm3. The covalently closed circular form has a sedimentation coefficient of 22S. These are 30 to 40 plasmid copies per genome equivalent, but growth in chloramphenicol results in amplification of the copy number to 600. In Proteus morganii 174, Mor174 coexists with a cryptic plasmid of molecular weight 15.8 X 10(6) and a buoyant density of 1.7170 g/cm3.     

盐生盐杆菌的质粒及其物理图谱

本文用４种不同的方法对１０株盐生盐杆菌是否含有质粒进行了检测,其中６株含有质粒,均属大质粒。几种检测方法中,以ＴＥＮＳ法效果好。Ｊ７菌株含有１种ＣＣＣ结构十分稳定的质粒,称之为ＰＨＨ２０５,其分子量较小,约为１６．７ｋｂ;拷贝数较高,为４９个／每个细胞。该粒在宿主的对数生长后期出现拷贝数高峰。通过限制性酶切分析,确定了５种酶在ＰＨＨ２０５上的切点,其中ＢａｍＨＩ,ＨｉｎｄＩＩＩ和ＸｂａＩ３种酶为单     

Temperature-dependent and amber copy mutants of plasmid R1drd-19 in Escherichia coli.

The isolation of conditional mutants with an altered copy number of the R plasmid R1drd-19 is described. Temperature-dependent as well as amber-suppressible mutants were found. These mutant plasmids have been named pKN301 and pKN303, respectively. Both types of mutations reside on the R plasmid. No difference in molecular weight could be detected by neutral sucrose gradient centrifugation for any of the mutant plasmids when compared with the wild-type plasmid. The number of copies of the plasmids was determined by measurement of the specific activity of the R plasmid-mediated β-lactamase and by measurement of covalently closed circular (CCC) DNA in alkaline sucrose gradients and dye-CsCl density gradients. Below 34 °C the temperature-dependent mutant, pKN301, had the same copy number as the wild type, while this was four times that of the wild type above 37 °C. The amber mutant pKN303 had a copy number indistinguishable from that of the wild-type plasmid in a strain containing a strong amber suppressor and a copy number about five times that of the wild-type plasmid in a strain lacking an amber suppressor. In a strain containing a temperature-sensitive amber suppressor, the amber mutant's copy number increased with the decrease in amber suppressor activity. Thus, the existence of the temperature-dependent and the amber-suppressible R-plasmid copy mutants indicates that the system that controls the replication of plasmid R1drd-19 contains an element with a negative function and that this element is a protein.     

Analysis of a recessive plasmid copy number mutant: evidence for negative control of Col E1 replication.

The Col E1-derivative copy number mutant plasmid pOP1Δ6 has been used to investigate the control of plasmid replication. pOP1Δ6 normally exists at about 200 copies per chromosome, while the wild-type plasmid from which it was derived (pBGP120) exists at about 15 copies per chromosome. We have observed that in E. coli containing both pOP1Δ6 and pBGP120, the copy number of pOP1Δ6 is lowered to 4–6 copies per chromosome. Thus the mutation in pOP1Δ6 is recessive. The association between the two plasmids is stable in E. coli, indicating that incompatibility properties as well as replication control characteristics have been altered in pOP1Δ6. Co-residence of the unrelated plasmid pSC101 with pOP1Δ6 has no detectable effect on pOP1Δ6 copy number. These results suggest that a plasmid-specific, diffusible repressor may act negatively to control plasmid copy number, and that pOP1Δ6 produces a defective repressor or is altered in repressor synthesis. We have constructed in vitro a plasmid which is identical in size to pQP1Δ6 but contains a replication origin region derived from pBGP120. Since this plasmid, pNOP1, exists stably (like pBGP120) at 10–15 copies per chromosome, the high copy number of pOP1Δ6 is not related to its reduced size relative to pBGP120. To localize the mutation in pOP1Δ6 responsible for DNA overproduction, we have cloned fragments of pBGP120 into pOP1Δ6 and selected for plasmids with wild-type copy number. We find that a 2.0 kb region of pBGP120 DNA surrounding the origin of plasmid DNA replication is capable of suppressing the DNA overproducer phenotype of pOP1Δ6. The 2.0 kb fragment is capable of independent self-replication or can integrate into pOP1Δ6 in vivo to form a composite plasmid with two origins of replication. The overproducer phenotype of pOP1Δ6 is suppressed in either configuration.     

Nonintegrated plasmid-chromosome complexes in Escherichia coli.

A number of plasmid systems have been examined for the ability of their covalently closed circular deoxyribonucleic acid (CCC DNA) forms to cosediment in neutral sucrose gradients with the folded chromosomes of their respective hosts. Given that cosedimentation of CCC plasmid and chromosomal DNA represents a bound or complexed state between these replicons, our results can be expressed as follows. (i) All plasmid systems complex, on the average, at least one plasmid per chromosomal equivalent. (ii) Stringently controlled plasmids exist predominantly in the bound state, whereas the opposite is true for plasmids that exist in multiple copies or are under relaxed control of replication. (iii) The degree to which a plasmid population binds to host chromosomes appears to be a function of plasmid genotype and not of plasmid size. (iv) For the colicin E1 plasmid the absolute number of plasmids bound per folded chromosome equivalent does increase as the intracellular plasmid/chromosome ratio increases in cells starved for required amino acids or in cells treated with chloramphenicol; however, the ratio of bound to free plasmids remains constant during plasmid copy number amplification.     

Plasmid deoxyribonucleic acid in strains of Bacillus sphaericus and in Bacillus moritai

Bacillus moritai and six strains of Bacillus sphaericus pathogenic to dipteran larvae were examined for the presence of covalently closed circular (CCC) DNA. The plasmid profiles of the bacteria were analyzed using a cleared lysate electrophoresis technique. Four of the six strains of B. sphaericus examined contained CCC DNA. Strain SSII-1 contained two plasmids (pKA1, pKA2) having molecular weights of about 8.4 and 2.0 megadaltons (MDa). Strains 1404 and 1881 each contained one plasmid, pKA3 and pKA4, respectively. pKA3 had a molecular weight of about 8.2 MDa. pKA4 had a relatively large plasmid with a molecular weight of about 33.5 MDa. Strain K contained five size classes of CCC DNA. The plasmids pKA5, pKA6, pKA7, pKA8, and pKA9 had molecular weights of about 11.4, 10.9, 7.4, 7.0, and 6.4 MDa, respectively. Strains 1593-4 and 1691 were plasmidless and could not be distinguished from each other based on their plasmid profiles. B. moritai ATCC 21042 contained two size classes of CCC duplex DNA; pRF100 had a molecular weight of about 4.6 MDa and pRF101 had a molecular weight of about 2.1 MDa. No phenotype association with any of the isolated plasmids has been determined.     

New class of polyomavirus mutant that can persist as free copies in F9 embryonal carcinoma cells.

A small circular DNA was found extrachromosomally in a clone of F9 embryonal carcinoma (EC) cells at high copy numbers per cell. The DNA was cloned in plasmid pUC19. Restriction endonuclease analyses of the DNA indicated that the DNA (fPyF9) was a mutant of polyomavirus (Py) DNA and had a mutation in a noncoding regulatory region. There have been many reports on the isolation of Py mutants capable of replication in undifferentiated cells. However, fPyF9 was different from other Py mutants in the following aspects: it was harbored stably as a free copy at 1 X 10(4) to 5 X 10(4) copies per cell in EC cells; it replicated in undifferentiated cells better than in differentiated cells; it was extremely rearranged in the sequences of the enhancer B domain; and it carried in the enhancer B domain three copies of an exogenous sequence which does not exist in Py strain A2. From these observations, we propose a new class of Py EC mutant which has an autonomous state similar to that of plasmid and small circular DNA in host cells.     

Genetic dissection of centromere function.

A system to detect a minimal function of Saccharomyces cerevisiae centromeres in vivo has been developed. Centromere DNA mutants have been examined and found to be active in a plasmid copy number control assay in the absence of segregation. The experiments allow the identification of a minimal centromere unit, CDE III, independently of its ability to mediate chromosome segregation. Centromere-mediated plasmid copy number control correlates with the ability of CDE III to assemble a DNA-protein complex. Cells forced to maintain excess copies of CDE III exhibit increased loss of a nonessential artificial chromosome. Thus, segregationally impaired centromeres can have negative effects in trans on chromosome segregation. The use of a plasmid copy number control assay has allowed assembly steps preceding chromosome segregation to be defined.     

Sporulation-converting bacteriophages for Bacillus pumilus.

Thirty-three sporulation-converting bacteriophages for Bacillus pumilus NRS576 were assigned to two apparently unrelated groups on the basis of morphology and antiserum neutralization. Bacterial sporulation mutants responded similarly (conversion or nonconversion) to representatives of both phage groups. Evidence is presented indicating that PMB1 and related phages specify a restriction and/or modification system.     

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.     

R6K Plasmid Replication: Influence of Chromosomal Genotype in Minicell-Producing Strains of Escherichia coli K-12

Alkaline sucrose velocity sedimentation and cesium chloride-ethidium bromide equilibrium centrifugation have been used to determine the number of copies per chromosomal equivalent of the relaxedly replicating R6K plasmid (a conjugative plasmid conferring ampicillin and streptomycin resistance) in two minicell-producing strains of Escherichia coli K-12. In one strain, the average number of covalently closed circular R6K molecules per chromosomal equivalent is 13 in log-phase and 35 in stationary-phase cells. In the other strain, there is an average of six covalently closed circular R6K molecules per chromosomal equivalent in both log- and stationary-phase cells. Selection from this strain of spontaneously occurring mutants resistant to high concentrations of ampicillin has been accomplished and such mutants show a two- to threefold increase in the number of R6K copies per chromosomal equivalent. Relative to the parental strain, mutants display the following properties: (i) elevated streptomycin resistance, (ii) a 10-fold increase in R6K conjugal transfer, (iii) a 10-fold increase in the amount of R6K plasmid deoxyribonucleic acid segregated into minicells, and (iv) a two- to threefold increase in R6K-specified beta-lactamase. The mutation(s) responsible for the increase in the number of R6K molecules per chromosomal equivalent is located on the bacterial chromosome. No R6K-linked mutations conferring the above phenotypes have been obtained. The mutations are presumed to be in chromosomal genes which play a role in the regulation of R6K replication in this strain.