Isolation and Characterization of Plasmid DNA in Streptococcus cremoris

Nine industrially important strains of Streptococcus cremoris (HP, AM₂, ML₁, WC, C₃, R₁, E₈, KH, and Wg₂) were shown to possess a diversity of plasmid molecules. Molecular weights of plasmids were determined from their relative mobilities after agarose gel electrophoresis and via electron microscopy. To illustrate the varied plasmid sizes, strain HP contained plasmids of 26, 18, 8.5, 3.3, and 2 megadaltons (Mdal); strain ML₁ contained plasmids of 29, 18, 9, 4, 2.2, and 1.8 Mdal; and strain AM₂ had plasmids of 42, 27, 16, and 8.4 Mdal. The numbers of plasmids observed in the other strains were 6, 5, 5, 7, 5, and 4 for C₃, E₈, KH, R₁, WC, and Wg₂, respectively. A spontaneous proteinase-negative (Prt⁻) mutant of HP was missing the 8.5-Mdal plasmid, which suggests that in this strain proteinase activity could be linked to this particular plasmid. A lactose-negative (Lac⁻) Prt⁻ mutant of ML₁ lacked the 2.2-Mdal plasmid. Under the conditions employed, antibiotic sensitivity and heavy-metal susceptibility did not correlate with the missing plasmid in Prt⁻ HP or in the Lac⁻ Prt⁻ ML₁. Curing experiments with AM₂, using acridine dyes and elevated temperatures, did not yield Lac⁻ variants. AM₂ was also cultured at high dilution rates in a chemostat for 168 h by using a buffered milk or lactic broth at 18 or 32°C with no selection of Lac⁻ derivatives. The inability to obtain Lac⁻ variants under conditions known to facilitate plasmid elimination suggests that lactose metabolism is not plasmid-mediated in AM₂.     

Plasmid Profiles of Lactose-Negative and Proteinase-Deficient Mutants of Streptococcus lactis C10, ML3, and M18

Lactose- and proteinase-negative (Lac⁻ Prt⁻) mutants of Streptococcus lactis C10, ML3, and M18 were isolated after treatment with ethidium bromide. The Lac⁻ Prt⁻ mutants of C10 were missing a 40-megadalton plasmid. A 33-megadalton plasmid was absent in the ML3 mutants, and the M18 variants lacked a 45-megadalton plasmid. The results suggest a linkage of these metabolic traits to the respective plasmids. The possible complexity of the interrelationship between lactose metabolism and proteinase activity is presented.     

Stabilization of Lactose Metabolism in Streptococcus lactis C2

The integration of the lactose plasmid from lactic streptococci into the host chromosome could stabilize this trait for dairy fermentations. Sixty lactose-positive (Lac⁺) transductants of lactose- and proteinase-negative (Lac⁻ Prt⁻) LM0220 were induced for temperature phage by UV irradiation or mitomycin C. Four of the transductants, designated KB18, KB21, KB54, and KB58, yielded lysates demonstrating less than one Lac⁺ transductant per 0.2 ml of phage lysate. Successive transferring in the presence of acriflavine did not yield Lac⁻ segregants from KB18, KB21, KB54, or KB58, whereas Streptococcus lactis C2 (parent culture) and three other Lac⁺ transductants showed 12 to 88% conversion from Lac⁺ to Lac⁻ within 6 to 10 repetitive transfers. When grown in continuous culture, KB21 did not show any Lac⁻ variants in 168 h, while S. lactis C2 had 96% conversion from Lac⁺ to Lac⁻ in 144 h. Agarose gel electrophoresis of plasmid DNA isolated from KB18, KB21, KB54, and KB58 revealed that the lactose plasmid, pLM2103, normally present in Lac⁺ transductants, was missing. This suggested integration of the transferred lactose plasmid into the chromosome. In contrast to phage lysates induced from S. lactis C2, which exhibited an exponential decrease in the number of Lac⁺ transductants after exposure to small doses of UV irradiation, the transduction frequency for lactose metabolism was stimulated by UV irradiation of lysates from KB58. The latter indicated chromosomal linkage for lac and that integration of the lactose genes plasmid into the chromosome had occurred.     

Characterization of Phage-Sensitive Mutants from a Phage-Insensitive Strain of Streptococcus lactis: Evidence for a Plasmid Determinant that Prevents Phage Adsorption

A phage-insensitive strain of Streptococcus lactis, designated ME2, was used as a prototype strain for the study of mechanisms and genetics of phage resistance in the lactic streptococci. Mutants sensitive to a Streptococcus cremoris phage, ϕ18, were isolated at a level of 17% from cultures of ME2 after sequential transfer at 30°C. Phage-sensitive mutants of ME2 were not fully permissive to ϕ18. The efficiency of plating of ϕ18 on the mutants was 5 × 10⁻⁷ as compared with <10⁻⁹ for ϕ18 on ME2. Further characterization of the mutants showed that they efficiently adsorbed ϕ18 at levels of >99.8%, whereas ME2 adsorbed only 20 to 40% of ϕ18. These results suggest that increased phage susceptibility of the mutants may result from the loss of a mechanism that inhibits phage adsorption. Moreover, the high frequency of spontaneous mutation in ME2 indicates the involvement of an unstable genetic determinant in this phage defense mechanism. ME2 was shown to possess 13 plasmids ranging in size from 1.6 to 34 megadaltons. Of 40 mutants examined that had increased efficiencies of plating, all were missing a 30-megadalton plasmid, pME0030. These data suggest that pME0030 codes for a function that prevents phage adsorption. Further phenotypic characterization of the phage-sensitive mutants showed that some mutants were deficient in the ability to ferment lactose (Lac) and hydrolyze milk proteins (Prt). However, the Lac⁺ and Prt⁺ phenotype segregated independently of the phage-sensitivity phenotype. One phage-sensitive adsorption mutant, designated N1, was tested for susceptibility to 14 different phages. N1 showed increased capacity to adsorb 4 and to replicate 2 of these 14 phages, thereby indicating a phage resistance mechanism in ME2 that generalizes to phage interactions other than the specific ϕ18-ME2 phage-host interaction. These data provide evidence for a unique plasmid-linked phage defense mechanism in phage-insensitive strains of lactic streptococci.     

Construction of Streptococcus lactis subsp. lactis Strains with a Single Plasmid Associated with Mucoid Phenotype

Lactose-fermenting mucoid (Lac⁺ Muc⁺) variants of plasmid-free Streptococcus lactis subsp. lactis MG1614 were obtained by protoplast transformation with total plasmid DNA from Muc⁺S. lactis subsp. cremoris ARH87. By using plasmid DNA from these variants for further transformations followed by novobiocininduced plasmid curing, Lac⁻ Muc⁺ MG1614 strains containing only a single 30-megadalton plasmid could be constructed. This plasmid, designated pVS5, appeared to be associated with the Muc⁺ phenotype.     

The Virulence Plasmid of Salmonella typhimurium Is Self-Transmissible

Most isolates of Salmonella enterica serovar Typhimurium contain a 90-kb virulence plasmid. This plasmid is reported to be mobilizable but nonconjugative. However, we have determined that the virulence plasmid of strains LT2, 14028, and SR-11 is indeed self-transmissible. The plasmid of strain SL1344 is not. Optimal conjugation frequency requires filter matings on M9 minimal glucose plates with a recipient strain lacking the virulence plasmid. These conditions result in a frequency of 2.9 × 10⁻⁴ transconjugants/donor. Matings on Luria-Bertani plates, liquid matings, or matings with a recipient strain carrying the virulence plasmid reduce the efficiency by up to 400-fold. Homologs of the F plasmid conjugation genes are physically located on the virulence plasmid and are required for the conjugative phenotype.     

Function of the HVCN1 proton channel in airway epithelia and a naturally occurring mutation,M91T

Airways secrete considerable amounts of acid. In this study, we investigated the identity and the pH-dependent function of the apical H⁺ channel in the airway epithelium. In pH stat recordings of confluent JME airway epithelia in Ussing chambers, Zn-sensitive acid secretion was activated at a mucosal threshold pH of ∼7, above which it increased pH-dependently at a rate of 339 ± 34 nmol × h⁻¹ × cm⁻² per pH unit. Similarly, H⁺ currents measured in JME cells in patch clamp recordings were readily blocked by Zn and activated by an alkaline outside pH. Small interfering RNA–mediated knockdown of HVCN1 mRNA expression in JME cells resulted in a loss of H⁺ currents in patch clamp recordings. Cloning of the open reading frame of HVCN1 from primary human airway epithelia resulted in a wild-type clone and a clone characterized by two sequential base exchanges (452T>C and 453G>A) resulting in a novel missense mutation, M91T HVCN1. Out of 95 human genomic DNA samples that were tested, we found one HVCN1 allele that was heterozygous for the M91T mutation. The activation of acid secretion in epithelia that natively expressed M91T HVCN1 required ∼0.5 pH units more alkaline mucosal pH values compared with wild-type epithelia. Similarly, activation of H⁺ currents across recombinantly expressed M91T HVCN1 required significantly larger pH gradients compared with wild-type HVCN1. This study provides both functional and molecular indications that the HVCN1 H⁺ channel mediates pH-regulated acid secretion by the airway epithelium. These data indicate that apical HVCN1 represents a mechanism to acidify an alkaline airway surface liquid.     

Involvement of a Plasmid in Production of Ropiness (Mucoidness) in Milk Cultures by Streptococcus cremoris MS

Curing and genetic transfer experiments showed that lactose-fermenting ability (Lac⁺) and the ability to produce mucoidness in milk cultures (Muc⁺) in Streptococcus cremoris MS were coded on plasmids. The Lac⁺ phenotype was associated with a 75.8-megadalton plasmid, pSRQ2201. The Muc⁺ phenotype was associated with a 18.5-megadalton plasmid, pSRQ2202. The Lac plasmid, pSRQ2201, was first conjugatively transferred from S. cremoris MS to Lac⁻S. lactis ML-3/2.2. Later, the Muc plasmid, pSRQ2202, was conjugatively transferred from Lac⁻ Muc⁺S. cremoris MS04 to Lac⁺ nonmucoid S. lactis transconjugant ML-3/2.201. Subsequently, pSRQ2201 and pSRQ2202 were cotransferred from Lac⁺ Muc⁺S. lactis transconjugant ML-3/2.202 to Lac⁻, nonmucoid, malty S. lactis 4/4.2 and S. lactis subsp. diacetylactis SLA3.25. Transconjugants showing pSRQ2201 were Lac⁺; those containing pSRQ2202 were Muc⁺. With the transfer of pSRQ2202, the transconjugants S. lactis ML-3/2.202 and S. lactis subsp. diacetylactis SLA3.2501 not only acquired the Muc⁺ phenotype but also resistance to bacteriophages, which were lytic to the respective parent strains S. lactis ML-3/2.201 and S. lactis subsp. diacetylactis SLA3.25.     

Studies on the Inoculation and Competitiveness of a Rhizobium leguminosarum Strain in Soils Containing Indigenous Rhizobia

The competitiveness of a Rhizobium leguminosarum strain was investigated at two separate locations in field inoculation studies on commercially grown peas. The soil at each location (sites I and II) contained an indigenous R. leguminosarum population of ca. 3 × 10⁴ rhizobia per g of soil. At site I it was necessary to use an inoculum concentration as large as 4 × 10⁷ CFU ml⁻¹ (2 × 10⁶ bacteria seed⁻¹) to establish the inoculum strain in the majority of nodules (73%). However, at site II the inoculum strain formed only 33% of nodules when applied at this (10⁷ CFU ml⁻¹) level. Establishment could not be further improved by increasing the inoculum concentration even as high as 10⁹ CFU ml⁻¹ (9.6 × 10⁷ bacteria seed⁻¹). The inoculum strain could be detected at both sites 19 months after inoculation. Analysis by intrinsic antibiotic resistance patterns and plasmid DNA profiles indicated that a dominant strain(s) and plasmid pool existed among the indigenous population at site II. Competition experiments were carried out under laboratory conditions between a dominant indigenous isolate and the inoculum strain. Both strains were shown to be equally competitive.     

Transfer of the Pea Symbiotic Plasmid pJB5JI in Nonsterile Soil

Transfer of the pea (Pisum sativum L.) symbiotic plasmid pJB5JI between strains of rhizobia was examined in sterile and nonsterile silt loam soil. Sinorhizobium fredii USDA 201 and HH003 were used as plasmid donors, and symbiotic plasmid-cured Rhizobium leguminosarum 6015 was used as the recipient. The plasmid was carried but not expressed in S. fredii strains, whereas transfer of the plasmid to R. leguminosarum 6015 rendered the recipient capable of nodulating pea plants. Confirmation of plasmid transfer was obtained by acquisition of plasmid-encoded antibiotic resistance genes, nodulation of pea plants, and plasmid profiles. Plasmid transfer in nonsterile soil occurred at frequencies of up to 10⁻⁴ per recipient and appeared to be highest at soil temperatures and soil moisture levels optimal for rhizobial growth. Conjugation frequencies were usually higher in sterile soil than in nonsterile soil. In nonsterile soil, transconjugants were recovered only with strain USDA 201 as the plasmid donor. Increasing the inoculum levels of donor and recipient strains up to 10⁹ cells g of soil⁻¹ increased the number of transconjugants; peak plasmid transfer frequencies, however, were found at the lower inoculum level of 10⁷ cells g of soil⁻¹. Plasmid transfer frequencies were raised in the presence of the pea rhizosphere or by additions of plant material. Transconjugants formed by the USDA 201(pJB5JI) × 6015 mating in soil formed effective nodules on peas.     

Viral Events Necessary for the Induction of Interferon in Chick Embryo Cells

Temperature-sensitive mutants of Sindbis virus were employed to investigate the nature of the viral event(s) which induces chick-embryo cells to produce interferon. Chick embryo cells induced by the parental heat-resistant strain of Sindbis virus produced essentially equal amounts of interferon at 29 and 42 C. An RNA⁻ and three RNA⁺ strains [temperature-sensitive mutants unable (RNA⁻) and able (RNA⁺) to make ribonucleic acid] produced interferon at 29 C but not at 42 C. It is concluded that viral RNA per se and the replication of viral RNA do not induce interferon production by chick embryo cells.     

Nature of the genetic determinant controlling exfoliative toxin production in Staphylococcus aureus

Phage group II Staphylococcus aureus has been identified as the etiological agent of the staphylococcal scaleded skin syndrome. The development of an animal model system permitted fulfillment of Koch's postulates and recognition of exfoliative toxin (ET) as being responsible for some of the clinical manifestations of this syndrome. Initial studies directed toward associating a lysogenic phage with the genetic control of ET synthesis failed to support this hypothesis. Growth of two Tox⁺ strains at 44 C was more effective than growth in ethidium bromide or sodium dodecyl sulfate in eliminating the ability to produce ET. The early and rapid accumulation of ET-negative (Tox⁻) variants during growth of strain UT 0007 at 44 C, the lack of any selective advantage of the Tox⁻ variants over Tox⁺ cells during growth at 44 C, and an enhanced elimination frequency at 44 C of 97.9% over the spontaneous frequency of loss strongly suggest that the gene for ET synthesis is extrachromosomal. Additional evidence suggests that this gene is located on a plasmid which is not associated with genes for penicillinase synthesis and cadmium resistance. Two Tox⁺ strains harbored lysogenic phage capable of transducing cadmium resistance, but not penicillin resistance, to specific Tox⁻ recipients.     

Bacterial Cell Division Regulation: Physiological Effects of Crystal Violet on Escherichia coli lon+ and lon− Strains

The Escherichia coli lon⁻ mutants apparently are defective in the ability to recommence cell division after temporary periods of deoxyribonucleic acid (DNA) synthesis inhibition. They are also more susceptible to cell division inhibition by the basic dye, crystal violet (CV), than are lon⁺ strains. In enriched broth, the lon⁺ strain continued to grow and divide in the presence of CV, but lon⁻ cell division was inhibited and filamentous growth resulted. In a supplemented minimal medium containing CV, lon⁻ cell division was only temporarily inhibited. There was no detectable specific effect on DNA synthesis, although CV slowed the rate of mass increase in both media. Trichloroacetic acid-insoluble lipid synthesis was preferentially inhibited in both lon⁺ and lon⁻ strains. In CV-containing enriched broth, diaminopimelic acid incorporation into trichloroacetic acid-insoluble compounds occurred at a rate greater than the rate of mass increase in both lon⁺ and lon⁻ strains. In a CV-containing supplemented minimal medium, diaminopimelic acid was incorporated to a greater extent by lon⁻ cells than by lon⁺ cells.     

Evidence for a Plasmid-Linked Restriction-Modification System in Lactobacillus helveticus

The presence of a restriction-modification (R/M) system against two bacteriophages, 328-B1 and hv, was demonstrated in three Lactobacillus helveticus strains, CNRZ 1094, CNRZ 1095, and CNRZ 1096. In addition, the burst size of phage 328-B1 in the three restrictive strains CNRZ 1094, CNRZ 1095, and CNRZ 1096 was reduced with respect to the values obtained in its propagating strain, CNRZ 328. Heating at 60°C did not inactivate the R/M system. Nonrestrictive variants from CNRZ 1094 were easily obtained under several culture conditions, but treatment with novobiocin at 42°C followed by storage at −20°C resulted in drastic elimination of the R⁺/M⁺ phenotype from all clones tested. Electrophoretic analysis of CNRZ 1094 nonrestrictive variants revealed the concomitant loss of a 34-kb plasmid. Four EcoRI fragments from the 34-kb plasmid were cloned in the Escherichia coli vector pACYC184. The use of one or several of these fragments as probes confirmed the plasmidic location of the genes responsible for the R/M system. These probes also showed the presence of R/M plasmids in the two other restrictive strains, CNRZ 1095 and CNRZ 1096. Lactose-fermenting ability and/or proteolytic capacity was not linked to the 34-kb plasmid.     

Growth Kinetics of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> ssp. <Emphasis Type="Italic">diacetylactis</Emphasis> Harboring Different Plasmid Content

The effect of plasmid content on growth of Lactococcus lactis ssp. diacetylactis harboring different plasmids and on plasmid stability was studied. Strain DRC-2C is a plasmid Lac⁺- and Prt⁺-free strain. Strain DRC-2 utilizes lactose as carbohydrate and has proteinase activity. The plasmid-free strain DRC-2C exhibited none of these features. Plasmid-encoded properties were clearly identified. Results showed that plasmid content decreased bacterial growth in terms of the specific growth rate determined. Slightly lower specific growth rate and lactic acid production were observed in the strain of higher plasmid content owing to the plasmid presence, causing metabolic burden to the host cell. The plasmid profile results showed that the number of bands in the two strains before and after fermentation were the same. This indicated that the plasmids were stably maintained and unchanged during the fermentation. Received: 27 July 2002 / Accepted: 27 August 2002     

R-Plasmid Transfer to and from Escherichia coli Strains Isolated from Human Fecal Samples

Strains of Escherichia coli recently isolated from human feces were examined for the frequency with which they accept an R factor (R1) from a derepressed fi⁺ strain of E. coli K-12 and transfer it to fecal and laboratory strains. Colicins produced by some of the isolates rapidly killed the other half of the mating pair; therefore, conjugation was conducted by a membrane filtration procedure whereby this effect was minimized. The majority of fecal E. coli isolates accepted the R factor at lower frequencies than K-12 F⁻, varying from 10⁻² per donor cell to undetectable levels. The frequencies with which certain fecal recipients received the R-plasmid were increased when its R⁺ transconjugant was either cured of the R1-plasmid and remated with the fi⁺ strain or backcrossed into the parental strain. The former suggests the loss of an incompatibility plasmid, and the latter suggests the modification of the R1-plasmid deoxyribonucleic acid (DNA). In general, the fecal R⁺E. coli transconjugants were less effective donors for K-12 F⁻ and heterologous fecal strains than was the fi⁺ K-12 strain, whereas the single strain of Citrobacter freundii examined was generally more competent. Passage of the R1-plasmid to strains of salmonellae reached mating frequencies of 10⁻¹ per donor cell when the recipient was a Salmonella typhi previously cured of its resident R-plasmid. However, two recently isolated strains of Salmonella accepted the R1-plasmid from E. coli K-12 R⁺ or the R⁺E. coli transconjugants at frequencies of 5 × 10⁻⁷ or less.     

Transposon Mutagenesis and Excision of R′ Plasmids by Conjugative, Chimeric Plasmid pUW942 in Extra-Slow-Growing Rhizobium japonicum Strains

Transposons Tn501 (specifying mercury resistance) and Tn7 (specifying resistance to trimethoprim and streptomycin) were introduced into extra-slow-growing Rhizobium japonicum by conjugal transfer of the 82 kilobase chimeric plasmid pUW942. Mercury-resistant transconjugants were obtained at a frequency of 10⁻⁷ to 10⁻⁹. The transfer frequency of streptomycin resistance was lower than that of mercury resistance, and Tn7 was relatively unstable. pUW942 was not maintained as an autonomously replicating plasmid in R. japonicum strains. However, some of the Hg^r transconjugants from the RJ19FY, RJ17W, and RJ12S strains acquired antibiotic markers of the vector plasmid pUW942. Southern hybridization of plasmid and chromosomal DNA of R. japonicum strains with ³²P-labeled pUW942 and pAS8Rep-1, the same plasmid as pUW942 except that it does not contain Tn501, revealed the formation of cointegrates between pUW942 and the chromosome of R. japonicum. More transconjugants with only Tn501 insertions in plasmids or the chromosome were obtained in crosses with strains RJ19FY and RJ17W than with RJ12S. These retained stable Hg^r both in plant nodules and under nonselective in vitro growth conditions. One of the RJ19FY and two of the RJ12S Hg^r transconjugants with vector plasmid-chromosome cointegrates conjugally transferred plasmids of 82, 84 or 86, and 90 kilobases, respectively, into plasmidless Escherichia coli C. These plasmids strongly hybridized to pUW942 and EcoRI digests of total DNA of each respective R. japonicum strain but not to indigenous plasmid DNA of the R. japonicum strains. These R′ plasmids consisted of pUW942-specific EcoRI fragments and an additional one or two new fragments derived from the R. japonicum chromosome.     

Relationship of the Presence and Copy Number of Plasmids to Exopolysaccharide Production and Symbiotic Effectiveness in Rhizobium fredii USDA 206

Rhizobium fredii USDA 206 harbors four large plasmids, one of which carries nodulation and nitrogen fixation genes. Previously isolated groups of plasmid-cured derivatives of strain USDA 206 were compared with each other to determine possible plasmid functions. Mutant strain 206CANS was isolated as a nonmucoid (Muc⁻) derivative of strain 206CA, a mutant that was cured of two plasmids. The Muc⁻ phenotype of 206CANS was only expressed when the strain was grown on certain media, particularly those with polyols as carbon sources. Plasmid pRj206b of strain 206CANS was previously shown to have a higher copy number than the same plasmid in strains USDA 206 and 206CA. When this plasmid was transferred to Muc⁺ strains, it conferred a nonmucoid phenotype on recipient strains. The symbiotic effectiveness of the wild-type and cured strains was compared. Overall, few differences were shown, but strains 206CA and 206CANS were found to have higher nitrogenase activities than the other strains. Thus, there appeared to be a possible relationship among exopolysaccharide synthesis, plasmid copy number, and symbiotic effectiveness.     

Isolation and Characterization of a Composite Plasmid Rms201 Mutant Temperature Sensitive for Replication

A mutant temperature-sensitive for R-plasmid replication, Rms201ts14, was isolated from composite plasmid Rms201 after mutagenesis of P1 transducing lysate with 100 mM hydroxylamine for 40 h at 37°C. When Escherichia coli ML1410(Rms201ts14)⁺ was grown at temperatures between 40 and 42°C in L broth, antibiotic-sensitive cells were segregated. When the incubation temperature of ML1410(Rms201ts14)⁺ in L-broth was shifted to 42 from 30°C, the increase in the number of antibiotic-resistant cells ceased 90 min after the temperature shift. However, the total number of cells continuously increased, and only 3% of the cells retained the plasmid at 5 h after the temperature shift to 42°C. At 30°C the amounts of covalently closed circular deoxyribonucleic acid per chromosome of Rms201ts14 and Rms201 were 3.8 and 6.3%, respectively. Incorporation of radioactive thymidine into the covalently closed circular deoxyribonucleic acid of Rms201ts14 did not take place at 42°C, whereas radioactive thymidine was incorporated into the covalently closed circular deoxyribonucleic acid of Rms201 at a rate of 4%/chromosome even at 42°C. The synthesis of plasmid covalently closed circular deoxyribonucleic acid in a cell harboring Rms201ts14 was almost completely blocked at 42°C. These results indicated that the gene(s) responsible for plasmid deoxyribonucleic acid replication was affected in the mutant Rms201ts14. Temperature-sensitive miniplasmid pMSts214, which has a molecular weight of 5.3 × 10⁶ and encodes ampicillin resistance, was isolated from Rms201ts14. Similarly, miniplasmid pMS201, which encodes single ampicillin resistance, was isolated from its parent, Rms201, and its molecular weight was 4.7 × 10⁶. These results indicate that the gene(s) causing temperature sensitivity for replication of Rms201 resides on the miniplasmid.