Plasmid-determined 2-hydroxypyridine utilization by Arthrobacter crystallopoietes.

Arthrobacter crystallopoietes has the ability to utilize 2-hydroxypyridine (2-HP) as a source of carbon and nitrogen and forms a blue extracellular pigment when grown in the presence of 2-HP. Ultracentrifugal analyses of pigment producing (Pig+) and pigment nonproducing (Pig-) strains of A. crystallopoietes revealed the presence of plasmid material in both strains. Recovery of plasmid DNA from Pig+ strains is two or three times greater than from Pig- strains. The molecular weight of plasmid DNA recovered from Pig+ strains (62 Mdaltons) is slightly higher than the molecular weight of plasmid DNA from Pig- strains. Consistent with the characterization of plasmid DNA from the two strains is that Pig+ strains contain a 63-Mdalton plasmid encoding 2-HP utilization as well as a cryptic plasmid of very nearly equal molecular weight. Pig- strains contain only the cryptic plasmid.     

Plasmid profile types of virulent and avirulent strains of phase-I Shigella sonnei and their rough phase-II and R-form variants

The study of S. sonnei in phase I, irrespective of their virulence, has revealed the existence of at least 3 types of profiles of large plasmids: (I)A having a single plasmid with a molecular weight of about 120 MD; (I)B having, alongside plasmid pSS120, a plasmid with a molecular weight of about 60 MD; (I)C, represented only by vaccine strain 6S, having three plasmids with molecular weights of about 80, 60 and 37 MD. The plasmid profiles of rough S. sonnei in phase II are characterized by the absence of large plasmids with a molecular weight of 120-80 MD, typical of bacteria in phase I, and can be in their turn subdivided, in accordance with the type of the initial culture, into three subvariants (II)A, (II)B and (II)C. The plasmid profiles of rough S. sonnei (R-forms and phase II) completely coincide. The biosynthesis of the specific antigen of S. sonnei in phase I can be determined by smaller derivatives obtained from large plasmid pSS120 by deletion (e.g., by a plasmid with a molecular weight of about 80 MD, such as plasmid pSS80).     

Effect of the molecular weight of DNA on the effectiveness of plasmid transformation of E. coli K12

The level of plasmid transformation and transfection by the high molecular mass DNA was studied for Escherichia coli mutants having increased efficiency of plasmid transformation by low molecular mass DNA. Decreased level of plasmid transformation and transfection registered in some mutants as compared to the one in wild type strain suggests the specificity of Escherichia coli cells penetration for DNA of different molecular mass.     

Method for the isolation of the replication region of a bacterial replicon: construction of a mini-F'kn plasmid.

A purified fragment of deoxyribonucleic acid (DNA) that determines resistance to kanamycin and is incapable of self-replication was used to select a self-replicating fragment from an EcoRI endonuclease digest of the sex factor F'lac. This F'lac fragment, exhibiting a molecular weight of 6 X 10(6), carries the genes essential for maintenance of the F replicon in Escherichia coli cells. The constructed mini-F'km plasmid also retains the incompatibility properties of the parent F'lac plasmid. Large amounts of the kanamycin resistance fragment of a molecular weight of 4.5 X 10(6) with an EcoRI-cleaved, self-replicating derivative of colicinogenic plasmid E1 that has a molecular weight of 2.2 X 10(6), The recombinant plasmid is able to replicate extensively in E. coli in medium containing chloramphenicol, and, therefore, large quantities of this plasmid DNA can be obtained. The substantial difference in size between the two fragments in the recombinant plasmid greatly facilitates their separation by preparative agarose gel electrophoresis.     

Plasmid pAP20 controlling the hemolytic activity of E. coli

The authors investigated pAP20 plasmid identified in E. coli cells isolated from man. According to the evidence obtained pAP20 plasmid determines the synthesis of alpha-hemolysin, being an F-like plasmid of the drd type. Having medium molecular size, the plasmid belongs to the inc FIV group and is partly incompatible with pAP38 plasmid which is a reference plasmid of the inc FVII group.     

SPP1-mediated plasmid transduction.

The virulent Bacillus subtilis phage SPP1 transduces plasmid DNA. Plasmid-transducing phages contain only plasmid DNA. Such DNA represents a concatemer of monomeric plasmid molecules with the molecular weight of mature SPP1 DNA. Biological parameters of plasmid transduction are described.     

微量法鼠疫菌质粒DNA抽提的优化

旨在分析微量法抽提鼠疫菌质粒DNA的效果,探讨其在鼠疫菌分子生物学实验研究中的应用价值.采用微量法分别提取鼠疫菌EV76株,假结核耶尔森菌PstII株及大肠杆菌V517株质粒DNA,琼脂糖凝胶电泳对质粒DNA抽提结果进行分析.结果显示,微量法能在较短时间内获取开环较少的闭合环状鼠疫菌质粒DNA,经琼脂糖凝胶电泳图示其电泳条带清晰、亮度均一.微量法鼠疫菌质粒DNA抽提效率和纯度较好,抽提结果稳定,重复性良好.经微量法抽提的质粒DNA符合多数鼠疫菌分子生物学试验的要求,可广泛应用于鼠疫菌分子生物学试验研究中.     

Enzymatic purification of plasmid DNA.

A novel method for plasmid DNA purification using enzymes that degrade all major types of contaminating nucleic acids present in crude plasmid DNA mixtures (but not plasmid DNA) has been devised. This method is quick (can be accomplished in two hours), requires no expensive laboratory equipment (an ultracentrifuge is not necessary) and is inexpensive. Plasmid DNA purified by this methodology can be used in a variety of molecular biological techniques including restriction enzyme digestions, subcloning, sequencing, nick translation and end labeling. This plasmid purification technique will be very useful for the molecular biologist performing cloning experiments.     

Biochemical Studies of Two Bacillus pumilus Plasmids

Bacillus pumilus NRS 576 harbored an estimated two copies per chromosome of a covalently closed, circular (CCC) deoxyribonucleic acid (DNA) molecule, the 576 plasmid. The 576 plasmid has a buoyant density of 1.698 g/cm(3) and a molecular weight of about 28 x 10(6). Plasmid copy number remained about the same in both exponentially growing and stationary-phase cells. Spontaneous variants of NRS 576 that formed spores at an elevated frequency were designated as W mutants. W mutants appeared to have lost the 576 plasmid on the basis of the following: W mutants (38 tested) lacked detectable CCC DNA, and the majority of the plasmid homologous sequences in bulk NRS 576 DNA were absent from bulk W mutant DNA. B. pumilus ATCC 7065 harbored at least 10 copies per chromosome of a CCC DNA element, the 7065 plasmid. The 7065 plasmid has a buoyant density of 1.696 g/cm(3) and a molecular weight of about 6 x 10(6). Although the copy number of the plasmid appeared to remain the same in exponentially growing and stationary-phase cells, an additional CCC form of higher molecular weight was detected in stationary-phase cells.     

人类泡沫逆转录病毒

根据 pHSRV1 3全基因组图谱和 pBR3 2 2质粒载体上限制酶切位点的性质 ,利用分子克隆技术 ,使 pHSRV1 3质粒上的bel1基因缺失突变并重新构建质粒载体 pHSRV1 3 -BR3 2 2 ,最后克隆到 1株含有目的基因组片段的重组质粒。     

Wide distribution of a 36-megadalton plasmid among clinical and environmental Spanish isolates ofLegionella pneumophila serogroup 1

With the eventual goal of characterizingLegionella pneumophila serogroup 1 plasmids at the molecular level, we have analyzed the plasmid contents of 78 clinical and environmental Spanish isolates. After selection of a suitable alkaline lysis method, we detected plasmids with approximate molecular weights of 25, 36, 40, 61, 80, 85, 90, and 95 megadalton (MDal). Several factors (i.e., wide temporal and geographic distribution, high frequency in both clinical and environmental isolates, and apparent high copy number after subculturing) make the 36 MDal type IA plasmid an appropriate plasmid for further molecular studies.     

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).     

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.     

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.     

Physicochemical properties of the pKMR plasmids controlling antibiotic resistance and the capacity to produce colonization antigen

KMR plasmids controlling antibiotic resistance and the capacity for production of the colonization antigen were identified in wild strains of E. coli (026, 0126, 0124) and S. sonnei isolated from patients with acute intestinal diseases. The strains of E. coli 026 and E. coli 0126 carried p KMR207-1 plasmid determining resistance to chloramphenicol and tetracycline and the adhesive properties. The molecular weight of the plasmid is 98 mD. The strain of S. sonnei carried p KMR 208-1 plasmid responsible for resistance to streptomycin, chloramphenicol and tetracycline and the adhesive properties. The molecular weight of this plasmid is 98 mD. The resistance to streptomycin and tetracycline and the capacity for the synthesis of the colonization antigen in E. coli 0214 was controlled by p KMR209 plasmid with the molecular weight of 2.66 mD. The restriction analysis suggests that p KMR207a-1 and p KMR 207b-1 plasmids detected in E. coli of different serotypes were identical, since they could be broken with BamH1 endonuclease into equal numbers of fragments similar by their molecular weights. p KMR207-1 and p KMR208-1 plasmids differed in their sensitivity to BamH-1 endonuclease. However, they were broken into 6 fragments similar by their molecular weights. p KMR207-1 and p KMR208-1 plasmids are probably closely related but not identical.     

Stability of recombinant plasmids on the continuous culture of Bifidobacterium animalis ATCC 27536

Bifidobacterium animalis ATCC 27536 represents among bifidobacteria a host-model for cloning experiments. The segregational and structural stabilities of a family of cloning vectors with different molecular weights but sharing a common core were studied in continuous fermentation of the hosting B. animalis without selective pressure. The rate of plasmid loss (R) and the specific growth rate difference (delta mu) between plasmid-free and plasmid-carrying cells were calculated for each plasmid and their relationship with plasmid size was studied. It was observed that both R and the numerical value of delta mu increased exponentially with plasmid size. The exponential functions correlating the specific growth rate difference and the rate of plasmid loss with the plasmid molecular weight were determined. Furthermore, the smallest of the plasmids studied, pLAV (4.3-kb) was thoroughly characterized by means of its complete nucleotide sequence. It was found that it contained an extra DNA fragment, the first bifidobacterial insertion sequence characterised, named IS 1999.     

Isolation, characterization and expression of a plasmid encoding chromate resistance in Pseudomonas putida KT2441

The chromium resistance properties encoded by a natural plasmid recovered from the environment were investigated. A 200 kb plasmid was isolated by the exogenous plasmid isolation method. The plasmid conferred a chromate resistance phenotype (MIC 8 mmol l⁻¹) to a chromate susceptible strain of Pseudomonas putida KT 2441 (MIC 0·5 mmol l⁻¹). The resistant strain took up 50% less ⁵¹Cr than the isogenic susceptible strain of Ps. putida KT2441. In addition, the resistant strain expressed two new membrane proteins encoded by the plasmid, an outer membrane protein (molecular weight 60 000) and an inner membrane protein (molecular weight 35 000). The physiological significance of these proteins is under current investigation.     

Identification of an Rts1 DNA fragment conferring temperature-dependent instability to vector plasmids

The multiphenotypic drug resistance factor Rts1 expresses a temperature-dependent instability characteristic. This plasmid was digested with the restriction enzyme BamHI. A DNA fragment with a molecular weight of 5.6 MDa (the H fragment) was inserted into plasmid pBR322 (pFK896) or into pSC105 (pYH156) at the BamHI site. These plasmids were unstable at 42 degrees C but stable at 32 degrees C. A restriction-enzyme map of the H fragment was constructed and the instability phenotype (Tdi) was localized to a DNA fragment with 0.5 MDa molecular weight. The temperature-dependent loss of the unstable plasmid pFK896 is abrupt and no gradual plasmid loss of this multicopy recombinant plasmid is observed. The possibility that the Tdi phenotype is due to overgrowth of R- cells was eliminated.