Transition of R factor NR1 in Proteus mirabilis: molecular structure and replication of NR1 deoxyribonucleic acid.

The structure of R factor NR1 DNA in Proteus mirabilis has been studied by using the techniques of CsCl density gradient centrifugation, sedimentation in neutral and alkaline sucrose gradients, and electron microscopy. It has been shown that the nontransitioned form of NR1 DNA isolated from P. mirabilis cultured in drug-free medium is a37-mum circular deoxyribonucleic acid (DNA) with a density of 1.712 g/ml in a neutral CsCl gradient. This circular molecule is a composite structure consisting of a 29-mum resistance transfer factor containing the tetracycline-resistance genes (RTF-TC) and an 8-mum r-determinants component conferring resistance to chloramphenicol (CM), streptomycin/spectinomycin, and the sulfonamides. There are one to two copies of NR1 per chromosome equivalent of DNA in exponential-phase cells cultured in Penassay broth. After growth of PM15/NR1 in medium containing 100 mug of CM per ml, the density of the NR1 DNA increased from 1.712 g/ml to approximately 1.718 g/ml and the proportion of NR1 DNA relative to the chromosome is amplified about 10-fold. The changes in R factor DNA structure which accompany this phenomenon (termed the transition) have been studied. DNA density profiles of the transitioned NR1 DNA consist of a 1.718 g/ml band which is skewed toward the less dense side. The transitioned NR1 DNA consists of molecules containing the RTF-TC element attached to multiple copies of r-determinants DNA (poly-r-determinant R factors) and multimeric and monomeric autonomous r-determinants structures. Poly-r-determinant R factors have a density intermediate between the basic composite structure (1.712 g/ml) and r-determinants DNA (1.718 g/ml). These species presumably account for the skewing of the 1.718-g/ml DNA band toward the less dense side. When transitioned cells are subsequently cultured in drug-free medium, poly-r-determinant R factors and autonomous poly-r-determinants undergo dissociation to form smaller structures containing fewer copies of r-determinants. This process continues until, after prolonged growth in drug-free medium the NR1 DNA returns to the nontransitioned state which consists of an RTF-TC and a single copy of r-determinants.     

Transition of the R Factor R12 in Proteus mirabilis

When Proteus mirabilis harboring the R factor R12 (a round of replication mutant of the R factor NR1) is cultured in medium containing streptomycin there can be an amplification in the number of copies of r-determinants per cell and the formation of enlarged polygenic R factors containing repeated sequences of r-determinants as well as polygenic molecules consisting of repeated sequences of r-determinants. This phenomenon has been referred to as the "transition." When transitioned cells are then cultured in drug-free medium, within a few generations two distinct density species of R factor deoxyribonucleic acid (DNA) are observed in a CsCl density gradient: a 1.712 g/ml band of covalently closed circular R factor DNA consisting of one transfer factor (RTF-TC) plus one r-determinant and a 1.718 g/ml band consisting of repeated sequences of r-determinants. The RTF-TC component of the R factor appears to control the replication of all the R factor DNA which is attached to it. In the autonomous state, however, polygenic sequences of r-determinants do not appear to replicate under the same control mechanism as when they are attached to an RTF-TC.     

EcoRI restriction endonuclease map of the composite R plasmid NR1.

A physical map of the composite R plasmid NR1 has been constructed using specific cleavage of deoxyribonucleic acid (DNA) by the restriction endonuclease EcoR-. Digestion of composite NR1 DNA by EcoRI yields thirteen fragments. The six largest fragments (designated A to F) are from the resistance transfer factor component that harbors the tetracycline resistance genes (RTF-TC). The seven smallest fragments (designated G to M) are from the r-determinants component that harbors the chloramphenicol (CM), streptomycin-spectinomycin (SM/SP), and sulfonamide (SA) resistance genes. The largest fragment of several RTF-TC segregants of NR1 that have deleted the r-determinants component is 0.8 X 10(6) daltons larger than fragment A of composite NR1. Only a part of fragment H of the r-determinants component is amplified in transitioned NR1 DNA in Proteus mirabilis, which consists of multiple, tandem sequences of r-determinants attached to a single copy of the RTF-TC component. Both of these changes can be explained by the locations of the excision sites at the RTF-TC: r-determinants junctions that are involved in the dissociation and reassociation of the RTF-TC and r-determinants components. The thirteen fragments of composite NR1 DNA produced by EcoRI have been ordered using partial digestion techniques. The order of the fragments is: A-D-C-E-F-B-H-I-L-K-G-M-J. The approximate locations of the TC, CM, SM/SP, and SA resistance genes on the EcoRI map were determined by analyzing several deletion mutants of NR1.     

Denaturation mapping of R factor deoxyribonucleic acid.

The R factor NR1 consists of two components: a resistance transfer factor which harbors the tetracycline resistance genes (RTF-TC) and the r-determinants component which harbors the other drug resistance genes. Using partial denaturation mapping it is possible to distinguish the RTF-TC region from the r-determinants region of the composite R factor NR1 DNA which has a contour length of 37 mum and a density of 1.712 g/ml. The r-determinants region was a relatively undenatured 8.5-mum segment of the molecule when the deoxyribonucleic acid was partially denatured at pH 10.7. An RTF-TC genetic segregant of NR1 which had lost the r-determinants component had a contour length of 28.7 mum and a density of 1.710 g/ml. Characterization of an RTF-TC using partial denaturation mapping at pH 10.7 confirmed that the relatively undenatured 8.5-mum r-determinants segment of the composite R factor had been deleted. Circular, transitioned NR1 DNA molecules (1.716 to 1.718 g/ml), whose contour lengths were consistent with an RTF-TC plus an integral number of tandem copies of r-determinants, were also characterized by denaturation mapping. The relatively undenatured region in these molecules had a length equal to an integral number of copies of r-determinants and was located at the same site in the partially denatured RTF-TC as the single copy of r-determinants in the 37-mum composite NR1. This indicates that there is a unique integration site for r-determinants in the RTF-TC component. The R factor UCR122, a TC deletion mutant of NR1, was also characterized by denaturation mapping. The translocation of the TC resistance gene(s) on the denaturation map permitted the alignment of the denaturation map with the heteroduplex map of Sharp et al. (u073). Linear and circular monomeric and presumed multimeric r-determinants DNA molecules (p = 1.718 g/ml) were partially denatured at a higher pH (11.10). The r-determinants multimers showed a repeating 8.3-mum (monomeric) partial denaturation pattern indicating a head-to-tail arrangement of monomers in these poly-r-determinant molecules.     

Drug Resistance of Enteric Bacteria

Drug resistance of 3,000 Shigella strains isolated in 1965 were investigated. These strains originated from 10 City Hospitals and 4 Prefectural Health Centers, which are located in different parts of Japan. One hundred and seventy strains which were resistant to 4 drugs, chloramphenicol (CM), tetracycline (TC), dihydrostreptomycin (SM), and sulfanilamide (SA), were selected at random from these stock cultures in this laboratory and the distribution of R factors in these isolates was examined. It was found that the strains all harbored R factors which were capable of transferring drug resistance by usual conjugal process. Among the strains carrying R factors, 85 per cent harbored a single type of R factor and 15 per cent carried two types of R factor in a cell. The latter is called the hetero-R state. Among the strains in the hetero-R state, isolation of strains harboring both R (SM.SA) and R (TC.CM.SM.SA) factors was most frequent. It was found that 25 R (SM.SA) factors isolated from strains in hetero-R had the genetic determinant iR^?, while most of the R (TC.CM.SM.SA) factors isolated from natural sources were iR⁺. When two types of R factor, R (SM.SA) and R (TC.CM.SM.SA) derived from the same host cells, were brought together in a host cell by superinfection with both factors, they were found to exist stably in a host bacterium. These results confirmed the stable existence of both factors in Shigella strains isolated from dysenteric patients.     

Transition of deletion mutants of the composite resistance plasmid NR1 in Escherichia coli and Salmonella typhimurium.

Derivatives of the composite R plasmid NR1 from which a portion of the resistance determinants (r-determinants) component had been deleted were found to undergo amplification of the remaining r-determinants region in Escherichia coli and Salmonella typhimurium. The wild-type NR1 plasmid does not amplify in these genera, although all of these plasmids undergo amplification in Proteus mirabilis. The deletion mutants retained the mercuric ion resistance operon (mer) but conferred a much lower level of sulfonamide resistance than NR1. The remaining r-determinants region, which is bounded by direct repeats of the insertion element IS1, formed multiple tandem duplications in E. coli, S. typhimurium, and P. mirabilis after subculturing the host cells in medium containing high concentrations of sulfonamide. Gene amplification was characterized by restriction endonuclease analysis, analytical buoyant density centrifugation, DNA-DNA hybridization, and sedimentation in sucrose gradients. The tandem repeats remained attached to the resistance transfer factor component of the plasmid in at least part of the plasmid population; autonomous tandem repeats of r-determinants were probably also present. Amplification did not occur in host recA mutants. Amplified strains subcultured in drug-free medium lost the amplified r-determinants. By using a strain temperature sensitive for the recA gene, it was possible to obtain gene amplification at the permissive temperature. Loss of r-determinants took place at the permissive temperature, but not at the nonpermissive temperature. The termini of the deletions of several independent mutants which conferred low sulfonamide resistance were found to be located within the adjacent streptomycin-spectinomycin resistance gene.     

Drug resistance of enteric bacteria. VII. Recombination of R factors with tetracycline-sensitive mutants

Hashimoto, Hajime (Gunma University, Maebashi, Japan), and Susumu Mitsuhashi. Drug resistance of enteric bacteria. VII. Recombination of R factors with tetracycline-sensitive mutants. J. Bacteriol. 92:1351-1356. 1966.-The transmissible drug-resistance factor R is able to confer resistance to tetracycline (TC), chloramphenicol (CM), streptomycin (SM), and sulfonamide (SA) on a host bacterium when infected by cell-to-cell contact. Tetracycline-sensitive mutants were isolated from either CM- or SM-sensitive mutants of an R factor. Among 30 mutants isolated, 10 were point mutants which could recombine with each other, forming recombinant R factors able to grow on plates containing 50 mug/ml of TC. The recombination frequency of TC-resistant recombinants was 10(-2) to 10(-3) in bacterial cells carrying two types of TC-sensitive R factors by superinfection with both factors. Segregational patterns of the various markers on the R factor, i.e., chl, str, sul, and m, the locus determining R mating, and their linkage order, were investigated among TC-resistant recombinants of the R factor. When TC was used as the selective drug, the tet locus mapped on the R factor as an end marker. In view of the fact that these results are inconsistent with the linkage order of various markers reported previously, a circular genetic structure for the R factor which includes five tet-s and three chl-s loci is presented.     

R-Factor-Mediated Resistance to Tetracycline in Proteus mirabilis

The expression of R-factor-mediated resistance to tetracycline has been compared in Proteus mirabilis and Escherichia coli. Resistance to a range of concentrations of tetracycline was significantly lower in P. mirabilis than in E. coli in both induced and repressed states. Indirect evidence showed that conditions which result in a marked increase in the level of resistance of P. mirabilis harboring the R factor NR1 to chloramphenicol, streptomycin, and spectinomycin due to an amplification in the number of copies of r-determinants per cell do not detectably increase the level of resistance to tetracycline. Tetracycline resistance was inducible in early stationary-phase P. mirabilis NR1 although not after 5 h in this state. Double isotope labeling of control and tetracycline-induced P. mirabilis NR1 in early stationary phase revealed isotopic enrichment of certain peaks in extracts from induced cells subjected to polyacrylamide gel electrophoresis.     

Unstable Mutants of R Factor

Thirty mutants sensitive to tetracycline were obtained from an R100 factor capable of conferring resistance to tetracycline (TC), chloramphenicol (CM), streptomycin (SM) and sulfanilamide (SA). Among the TC sensitive mutants, three showed a high frequency of spontaneous loss from host strains. The genetic loci governing the stability of R factor in host bacteria were denoted as stb. The stb^– R factors have lost many of the properties of a wild type R factor, such as, the capability to sexually transfer drug resistance and host chromosome, to confer superinfection immunity and to inhibit F function. All of these properties did not revert to a wild type phenotype, suggesting that these mutations are deletions including genetic determinants governing both TC resistance and stability of R factor. Recombinational analysis between stb^– and stb⁺ R factors indicated that crossovers between the stb loci and those governing CM (or SM.SA) resistance took place at high frequency. No crossovers were detected between stb loci and those governing TC resistance, indicating that the stb loci are linked closely to the loci governing TC resistance.     

Detection of R+ Bacteria in Cultured Marine Fish,Yellowtails (Seriola quinqueradiata)

Many drug-resistant gram-negative bacilli were isolated from the intestinal tracts of yellowtails (Seriola quinqueradiata) cultured on farms in various parts of Kochi and Ehime Prefectures, Shikoku Island. They were Vibrio, Pseudomonas, Proteus, Citrobacter and some unidentified Enterobacteriaceae. Of these drug-resistant strains, Vibrio and Pseudomonas were found to carry R factors in high frequencies. These R factors had four types of resistance markers, SA, SA. CM, SM. CM. ABP. and SA. SM. CM. TC. All R factors were found to belong to the fi^– type. In contrast, only one drug-resistant gram-negative bacillus was detected in a cultured yellowtail on farms near Numazu City, Shizuoka Prefecture.     

Gene recombination and segregation of resistance factor R in Escherichia coli

Hashimoto, Hajime (Osaka University, Osaka, Japan), and Yukinori Hirota. Gene recombination and segregation of resistance factor R in Escherichia coli. J. Bacteriol. 91:51-62. 1966.-Independent chloramphenicol-sensitive (CM(s)) mutants of the drug-resistance factor R were isolated. Introduction of two different R factor CM(s) mutants into a single bacterium, by conjugation or transduction, gave chloramphenicol-resistant (CM(r)) colonies when such strains were plated on a medium containing chloramphenicol (Cm). These CM(r) colonies resulted from recombination between two R factors contained within the same cell. Most of the CM(r) colonies were heterogeneous, and segregation of drug-resistance markers was observed among the progeny. Segregated bacteria which still carried the recombinant R factor were stable for resistance to Cm as well as for other markers of R. All the markers of recombinant R factors were cotransducible with high coincidence and at the same frequency as wild-type R. Sensitive mutants of R which had lost all the resistance markers of the R factor were found also. A mutation of R, referred to as SMA, which was sensitive to streptomycin and sulfanilamide, was capable of reverting to resistance to both of these drugs simultaneously. The sensitive alleles for SMA, CM, and TC were shown to be recessive to the resistance alleles. Mutants of R having multisite mutations or deletions in the CM gene were isolated and used to analyze the pattern of linked segregation of unselected markers of the recombinant R factor. The drug resistance factor R was shown to have two linkage groups, CM-SMA and TC-m.     

Molecular Recombination Between R-Factor Deoxyribonucleic Acid Molecules in Escherichia coli Host Cells

THREE PREVIOUSLY STUDIED R FACTORS WERE USED: 222/R4, controlling transmissible resistance to sulfonamide, streptomycin, chloromycetin, and tetracycline (SU(r) SM(r) CM(r) TC(r)); 222/R3, a derivative of 222/R4 (now termed 222/R3W) having lost TC(r); and R15, controlling infectious resistance to SU and SM only. Two types of derivative R factors were isolated from 222/R4 by serial subculture in Salmonella species. One derivative, termed 222/R1, lost resistance to SU, SM, and CM, and the other, termed 222/R3N, lost only TC(r). Each factor was transferred to a standard Escherichia coli K-12 host. Recombinant factors of 222/R4 phenotype were isolated by selection after mixed culture of E. coli (222/R1)(+) and (222/R3N)(+) strains. Density-gradient equilibrium centrifugation of lysates of E. coli R(+) hosts in the presence of ethidium bromide separated R-factor deoxyribonucleic acid (DNA) as a heavy satellite peak which was subjected to electron microscopy or analytical density gradient centrifugation. Each DNA comprised a unimolecular species of circular DNA. The contour of R15 measured 22.3 mum [equivalent to 46 x 10(6) atomic mass units (AMU)], and that of 222/R4 measured 33.6 mum (70 x 10(6) AMU). 222/R3W appeared to be a point mutant or small deletion of 222/R4 with an almost identical size, whereas 222/R3N had lost a DNA segment of about 3 mum, and measured 30.3 mum or 63 x 10(6) AMU. The 222/R1 factors also appeared to have arisen by loss of DNA from 222/R4, 222/R1A being 22.3 mum or 46 x 10(6) AMU, whereas all other 222/R1 factors appeared to be duplicates, measuring 25.6 mum or 53 x 10(6) AMU. The DNA from six recombinant factors of R4 phenotype was indistinguishable in size and configuration from the parental 222/R4. In most cases, the number of R-factor copies (present as covalently closed circular molecules) per copy of the E. coli chromosome was less than 2, ranging from 1.2 to 3.3.     

New Type of R Factors Incapable of Inactivating Chloramphenicol

Four R factors conferring chloramphenicol (CM) resistance were isolated from Escherichia coli strains of clinical origin. Strains carrying the factors were found to be incapable of inactivating the drug in the presence of acetyl coenzyme A. E. coli W3630 carrying R(70), one of these factors, became sensitive to CM after treatment with glycine, indicating that the spheroplasts of W3630 R(70) (+) were sensitive to the drug and suggesting that the cell membrane is important for CM resistance. The observation that cell-free protein synthesis in W3630 R(70) (+) was inhibited by CM is also compatible with a decrease in permeability. CM resistance in W3630 R(70) (+) appeared to be inducible, because (i) preincubation with subinhibitory concentrations of CM prevented the prolonged lag noted for growth in the presence of 25 mug of CM per ml, and (ii) the preincubation effect was lost after overnight growth in CM-free medium. By contrast, E. coli W3630 cml(+), in which the resistance determinant is integrated into the chromosome, was capable of rapid inactivation of CM. E. coli W3630 cml(+) R(70) (+), which contains the proposed permeability determinant (episomal) as well as levels of the inactivating enzyme (chromosomal) that are comparable with W3630 cml(+), was capable of brief inactivation of CM when inoculated into drug-containing medium. The absence of continued inactivation on more prolonged incubation favors the hypothesis that the R(70) factor inhibited further penetration of CM and that this property possesses the characteristics of induction.     

Drug Resistance of Enteric Bacteria

A nontransferable R₂₁ (TC) factor was obtained by transduction of R₁₀ (TC.CM.SM.SA) with phage epsilon in group E Salmonella. The R₂₁ (TC) factor acquired transmissibility by the normal conjugal process when group E Salmonella strains harboring R₂₁ (TC) factor were infected with wild-type F or R₁₆ (CM) factor. This transmissibility at high frequency was accounted for by the formation of the recombinant F TC and R₁₀ (CM) TC factors. The F TC and R₁₆ (CM) TC factors were genetically the same as the original F and R₁₆ (CM) factors, except for the ability to confer TC resistance. In the transduction of F TC factor with phage P1, a dF TC (d: defective) factor was obtained that was defective in many F properties, such as the ability to introduce host chromosome and produce male substance, but was capable of transducing TC resistance (dF TC-infection) at low frequency.     

Evaluation of culture systems containing bovine oviduct epithelial cells or granulosa cells to mature and maintain the developmental competence of bovine oocytes in vitro

The effects of estrous cow serum (ECS), bovine oviduct epithelial cells (BOEC), and bovine granulosa cells (GC) on in vitro maturation (IVM) of immature oocyte-cumulus complexes (OCCs) were evaluated. Selected OCCs were cultured for 24 to 26 h in microdroplets of culture medium (CM; TCM 199 + 25 mM HEPES + 100 mug gentamicin sulfate/ml) or in CM medium supplemented or conditioned with 20% ECS, BOEC +/- 20% ECS or GC + 20% ECS. Supplemented media were incubated for 2 h before addition of OCCs, whereas media were conditioned by incubation with 20% ECS or BOEC +/- 20% ECS for 6 d, or with 20% ECS +/- GC for 24 or 48 h before addition of OCCs. The developmental competence of oocytes after TVM was assessed by insemination with glass wool separated, frozen-thawed bovine spermatozoa in microdroplets of modified medium (TALP) containing heparin (5 mug/ml) and BOEC for 18 h. The presumptive zygotes were cultured in microdroplets of CM medium + 20% ECS + BOEC for 7 to 9 d to assess embryo development to morula and blastocyst stages. The percentages of OCCs undergoing IVM (85 to 94%) and in vitro fertilization (IVF) (66 to 80%) were high, irrespective of the IVM conditions. Only after the IVM of OCCs in CM medium alone was the percentage of oocytes undergoing IVF significantly lower (66%; P<0.05). The proportion of IVF oocytes developing to blastocysts with a normal complement of cells (126 to 138) increased significantly (P<0.05) when the OCCs were matured in supplemented or conditioned CM medium containing ECS and/or somatic cells (18 to 28%) compared with those in CM medium alone (9%). When the CM medium was supplemented or conditioned with GC + 20% ECS, the proportion of fertilized oocytes developing to blastocysts increased significantly (28%; P<0.05). These results indicate that the potential of immature OCCs to be fertilized and to complete embryonic development to the blastocyst stage in vitro is enhanced by maturation in CM medium containing 20% ECS and/or BOEC or GC.     

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.     

利用重组Pichia pastoris生产腺苷甲硫氨酸

为改造甲醇利用型酵母Pichia pastoris来生产腺苷甲硫氨酸（SAM,S－adenosyl-L-methionine),我们将一个带有SAM合成酶基因的胞内表达质粒转化入Pichia pastoris菌株GS115,经过G418抗性筛选得到一株有两个基因拷贝的转化子。该菌在含有甲醇和甲硫氨酸的培养基中生长5d后,其细胞内的SAM的产量比原始菌株提高了30余倍。对该菌生产SAM的培养基中的碳源与氮源进行了优化,结果显示碳源的控制对该菌SAM产量的影响很大。在试管水平,该菌在含有0．75％的L-methionine并且碳源和有机氮源经过一定程度优化的培养基中,生长6d后SAM产量达到1．58g/L。     

Drug resistance of Escherichia coli isolated in Nepal.

We collected Escherichia coli strains from 59 Nepalese porters in 1971 and surveyed for their drug resistance. Drug-resistant E. coli strains were isolated from four porters. (TC. CM. SM. SA. APC.)-resistant strains were isolated from two porters and SA- or APC-resistant strains were isolated from each of the others. The R factors were demonstrated from the multiple-resistant E. coli strains.     

Integration of Chloramphenicol Resistance Gene of an R Factor on Escherichia coli Chromosome

An unstable mutant R factor conferring only chloramphenicol (CM) resistance was obtained by spontaneous segregation. After storage in broth culture, a stable CM-resistant mutant was obtained and its CM-resistance could not be cured by treatment with acriflavine or transduced to a recombination-deficient strain of Escherichia coli K12. Recombinational analysis indicated that the cml gene governing CM resistance had been integrated into the E. coli chromosome and closely linked with met B locus. The cml gene was co-transduced with both met and arg markers by phage P1, and the linkage order was considered to be mtl-cml-met-arg-thi. When the strain carrying this chromosomal CM-resistance was infected with a transferable R (TC) factor capable of conferring tetracycline (TC) resistance, the CM-resistance became transferable by conjugation. This mechanism is considered to account for the formation of the recombinant R (TC.CM) factor.