Identification and Application of Plasmids Suitable for Transfer of Foreign DNA to Members of the Genus Gordonia

Gene transfer systems for Gordonia polyisoprenivorans strains VH2 and Y2K based on electroporation and conjugation, respectively, were established. Several parameters were optimized, resulting in transformation efficiencies of >4 × 10⁵ CFU/μg of plasmid DNA. In contrast to most previously described electroporation protocols, the highest efficiencies were obtained by applying a heat shock after the intrinsic electroporation. Under these conditions, transfer and autonomous replication of plasmid pNC9503 was also demonstrated to proceed in G. alkanivorans DSM44187, G. nitida DSM44499^T, G. rubropertincta DSM43197^T, G. rubropertincta DSM46038, and G. terrae DSM43249^T. Conjugational plasmid DNA transfer to G. polyisoprenivorans resulted in transfer frequencies of up to 5 × 10⁻⁶ of the recipient cells. Recombinant strains capable of polyhydroxyalkanoate synthesis from alkanes were constructed.     

Ultrasound-mediated DNA transfer for bacteria

In environmental microbiology, the most commonly used methods of bacterial DNA transfer are conjugation and electroporation. However, conjugation requires physical contact and cell–pilus–cell interactions; electroporation requires low-ionic strength medium and high voltage. These limitations have hampered broad applications of bacterial DNA delivery. We have employed a standard low frequency 40 kHz ultrasound bath to successfully transfer plasmid pBBR1MCS2 into Pseudomonas putida UWC1, Escherichia coli DH5α and Pseudomonas fluorescens SBW25 with high efficiency. Under optimal conditions: ultrasound exposure time of 10 s, 50 mM CaCl₂, temperature of 22°C, plasmid concentration of 0.8 ng/µl, P. putida UWC1 cell concentration of 2.5 × 10⁹ CFU (colony forming unit)/ml and reaction volume of 500 µl, the efficiency of ultrasound DNA delivery (UDD) was 9.8 ± 2.3 × 10⁻⁶ transformants per cell, which was nine times more efficient than conjugation, and even four times greater than electroporation. We have also transferred pBBR1MCS2 into E. coli DH5α and P. fluorescens SBW25 with efficiencies of 1.16 ± 0.13 × 10⁻⁶ and 4.33 ± 0.78 × 10⁻⁶ transformants per cell, respectively. Low frequency UDD can be readily scaled up, allowing for the application of UDD not only in laboratory conditions but also on an industrial scale.     

PVv3, a New Shuttle Vector for Gene Expression in Vibrio vulnificus

An efficient electroporation procedure for Vibrio vulnificus was designed using the new cloning vector pVv3 (3,107 bp). Transformation efficiencies up to 2 × 10⁶ transformants per μg DNA were achieved. The vector stably replicated in both V. vulnificus and Escherichia coli and was also successfully introduced into Vibrio parahaemolyticus and Vibrio cholerae. To demonstrate the suitability of the vector for molecular cloning, the green fluorescent protein (GFP) gene and the vvhBA hemolysin operon were inserted into the vector and functionally expressed in Vibrio and E. coli.     

A novel replicating circular DNAzyme

10–23 DNAzyme has the potential to suppress gene expressions through sequence-specific mRNA cleavage. However, the dependence on exogenous delivery limits its applications. The objective of this work is to establish a replicating DNAzyme in bacteria using a single-stranded DNA vector. By cloning the 10–23 DNAzyme into the M13mp18 vector, we constructed two circular DNAzymes, C-Dz₇ and C-Dz₄₈₂, targeting the β-lactamase mRNA. These circular DNAzymes showed in vitro catalytic efficiencies (k_cat/K_M) of 7.82 × 10⁶ and 1.36 × 10⁷ M^–1·min^–1, respectively. Their dependence on divalent metal ions is similar to that found with linear 10–23 DNAzyme. Importantly, the circular DNAzymes were not only capable of replicating in bacteria but also exhibited high activities in inhibiting β-lactamase and bacterial growth. This study thus provides a novel strategy to produce replicating DNAzymes which may find widespread applications.     

Application of the shsp Gene, Encoding a Small Heat Shock Protein, as a Food-Grade Selection Marker for Lactic Acid Bacteria

Plasmid pSt04 of Streptococcus thermophilus contains a gene encoding a protein with homology to small heat shock proteins (A. Geis, H. A. M. El Demerdash, and K. J. Heller, Plasmid 50:53-69, 2003). Strains cured from the shsp plasmids showed significantly reduced heat and acid resistance and a lower maximal growth temperature. Transformation of the cloned shsp gene into S. thermophilus St11 lacking a plasmid encoding shsp resulted in increased resistance to incubation at 60°C or pH 3.5 and in the ability to grow at 52°C. A food-grade cloning system for S. thermophilus, based on the plasmid-encoded shsp gene as a selection marker, was developed. This approach allowed selection after transfer of native and recombinant shsp plasmids into different S. thermophilus and Lactococcus lactis strains. Using a recombinant plasmid carrying an erythromycin resistance (Em^r) gene in addition to shsp, we demonstrated that both markers are equally efficient in selecting for plasmid-bearing cells. The average transformation rates in S. thermophilus (when we were selecting for heat resistance) were determined to be 2.4 × 10⁴ and 1.0 × 10⁴ CFU/0.5 μg of DNA, with standard deviations of 0.54 × 10⁴ and 0.32 × 10⁴, for shsp and Em^r selection, respectively. When we selected for pH resistance, the average transformation rates were determined to be 2.25 × 10⁴ and 3.8 × 10³ CFU/0.5 μg of DNA, with standard deviations of 0.63 × 10⁴ and 3.48 × 10³, for shsp and Em^r selection, respectively. The applicability of shsp as a selection marker was further demonstrated by constructing S. thermophilus plasmid pHRM1 carrying the shsp gene as a selection marker and the restriction-modification genes of another S. thermophilus plasmid as a functional trait.     

High-Frequency Phage-Mediated Gene Transfer among Escherichia coli Cells, Determined at the Single-Cell Level

Recent whole-genome analysis suggests that lateral gene transfer by bacteriophages has contributed significantly to the genetic diversity of bacteria. To accurately determine the frequency of phage-mediated gene transfer, we employed cycling primed in situ amplification-fluorescent in situ hybridization (CPRINS-FISH) and investigated the movement of the ampicillin resistance gene among Escherichia coli cells mediated by phage at the single-cell level. Phages P1 and T4 and the newly isolated E. coli phage EC10 were used as vectors. The transduction frequencies determined by conventional plating were 3 × 10⁻⁸ to 2 × 10⁻⁶, 1 × 10⁻⁸ to 4 × 10⁻⁸, and <4 × 10⁻⁹ to 4 × 10⁻⁸ per PFU for phages P1, T4, and EC10, respectively. The frequencies of DNA transfer determined by CPRINS-FISH were 7 × 10⁻⁴ to 1 × 10⁻³, 9 × 10⁻⁴ to 3 × 10⁻³, and 5 × 10⁻⁴ to 4 × 10⁻³ for phages P1, T4, and EC10, respectively. Direct viable counting combined with CPRINS-FISH revealed that more than 20% of the cells carrying the transferred gene retained their viabilities. These results revealed that the difference in the number of viable cells carrying the transferred gene and the number of cells capable of growth on the selective medium was 3 to 4 orders of magnitude, indicating that phage-mediated exchange of DNA sequences among bacteria occurs with unexpectedly high frequency.     

Electrotransformation of Clostridium thermocellum

Electrotransformation of several strains of Clostridium thermocellum was achieved using plasmid pIKm1 with selection based on resistance to erythromycin and lincomycin. A custom-built pulse generator was used to apply a square 10-ms pulse to an electrotransformation cuvette consisting of a modified centrifuge tube. Transformation was verified by recovery of the shuttle plasmid pIKm1 from presumptive transformants of C. thermocellum with subsequent PCR specific to the mls gene on the plasmid, as well as by retransformation of Escherichia coli. Optimization carried out with strain DSM 1313 increased transformation efficiencies from <1 to (2.2 ± 0.5) × 10⁵ transformants per μg of plasmid DNA. Factors conducive to achieving high transformation efficiencies included optimized periods of incubation both before and after electric pulse application, chilling during cell collection and washing, subculture in the presence of isoniacin prior to electric pulse application, a custom-built cuvette embedded in an ice block during pulse application, use of a high (25-kV/cm) field strength, and induction of the mls gene before plating the cells on selective medium. The protocol and preferred conditions developed for strain DSM 1313 resulted in transformation efficiencies of (5.0 ± 1.8) × 10⁴ transformants per μg of plasmid DNA for strain ATCC 27405 and ~1 × 10³ transformants per μg of plasmid DNA for strains DSM 4150 and 7072. Cell viability under optimal conditions was ~50% of that of controls not exposed to an electrical pulse. Dam methylation had a beneficial but modest (7-fold for strain ATCC 27405; 40-fold for strain DSM 1313) effect on transformation efficiency. The effect of isoniacin was also strain specific. The results reported here provide for the first time a gene transfer method functional in C. thermocellum that is suitable for molecular manipulations involving either the introduction of genes associated with foreign gene products or knockout of native genes.     

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.     

Anaerobic Transfer of Antibiotic Resistance from Pseudomonas aeruginosa

Pseudomonas aeruginosa, an opportunistic pathogen that often initiates infections from a reservoir in the intestinal tract, may donate or acquire antibiotic resistance in an anaerobic environment. Only by including nitrate and nitrite in media could antibiotic-resistant and -sensitive strains of P. aeruginosa be cultured in a glove box isolator. These anaerobically grown cells remained sensitive to lytic phage isolated from sewage. After incubation with a phage lysate derived from P. aeruginosa 1822, anaerobic transfer of antibiotic resistance to recipients P. aeruginosa PS8EtBr and PS8EtBrR occurred at frequencies of 6.2 × 10⁻⁹ and 5.0 × 10⁻⁸ cells per plaque-forming unit, respectively. In experiments performed outside the isolator, transfer frequencies to PS8EtBr and PS8EtBrR were higher, 1.3 × 10⁻⁷ and 6.5 × 10⁻⁸ cells per plaque-forming unit, respectively. When P. aeruginosa 1822 was incubated aerobically with Escherichia coli B in medium containing nitrate and nitrite, the maximum concentration of carbenicillin-resistant E. coli B reached 25% of the total E. coli B population. This percentage declined to 0.01% of the total E. coli B population when anaerobically grown P. aeruginosa 1822 and E. coli B were combined and incubated in the glove box isolator. The highest concentration of the recipient population converted to antibiotic resistance occurred after 24 h of aerobic incubation, when an initially high donor/recipient ratio (>15) of cells was mixed. These data indicate that transfer of antibiotic resistance either by transduction between Pseudomonas spp. or by conjugation between Pseudomonas sp. and E. coli occurs under strict anaerobic conditions, although at lower frequencies than under aerobic conditions.     

Plasmids pJP4 and r68.45 Can Be Transferred between Populations of Bradyrhizobia in Nonsterile Soil

IncP plasmid r68.45, which carries several antibiotic resistance genes, and IncP plasmid pJP4, which contains genes for mercury resistance and 2,4-dichlorophenoxyacetic acid degradation, were evaluated for their ability to transfer to soil populations of rhizobia. Transfer of r68.45 was detected in nonsterile soil by using Bradyrhizobium japonicum USDA 123 as the plasmid donor and several Bradyrhizobium sp. strains as recipients. Plasmid transfer frequencies ranged up to 9.1 × 10^-5 in soil amended with 0.1% soybean meal and were highest after 7 days with strain 3G4b4-RS as the recipient. Transconjugants were detected in 7 of 500 soybean nodules tested, but the absence of both parental strains in these nodules suggests that plasmid transfer had occurred in the soil, in the rhizosphere, or on the root surface. Transfer of degradative plasmid pJP4 was also evaluated in nonsterile soil by using B. japonicum USDA 438 as the plasmid donor and several Bradyrhizobium sp. strains as recipients. Plasmid pJP4 was transferred only when strains USDA 110-ARS and 3G4b4-RS were the recipients. The plasmid transfer frequency was highest for strain 3G4b4-RS (up to 7.4 × 10^-6). Mercury additions to soil, ranging from 10 to 50 μg/g of soil, did not affect population levels of parental strains or the plasmid transfer frequency.     

Ultra-Low Background DNA Cloning System

Yeast-based in vivo cloning is useful for cloning DNA fragments into plasmid vectors and is based on the ability of yeast to recombine the DNA fragments by homologous recombination. Although this method is efficient, it produces some by-products. We have developed an “ultra-low background DNA cloning system” on the basis of yeast-based in vivo cloning, by almost completely eliminating the generation of by-products and applying the method to commonly used Escherichia coli vectors, particularly those lacking yeast replication origins and carrying an ampicillin resistance gene (Amp^r). First, we constructed a conversion cassette containing the DNA sequences in the following order: an Amp^r 5′ UTR (untranslated region) and coding region, an autonomous replication sequence and a centromere sequence from yeast, a TRP1 yeast selectable marker, and an Amp^r 3′ UTR. This cassette allowed conversion of the Amp^r-containing vector into the yeast/E. coli shuttle vector through use of the Amp^r sequence by homologous recombination. Furthermore, simultaneous transformation of the desired DNA fragment into yeast allowed cloning of this DNA fragment into the same vector. We rescued the plasmid vectors from all yeast transformants, and by-products containing the E. coli replication origin disappeared. Next, the rescued vectors were transformed into E. coli and the by-products containing the yeast replication origin disappeared. Thus, our method used yeast- and E. coli-specific “origins of replication” to eliminate the generation of by-products. Finally, we successfully cloned the DNA fragment into the vector with almost 100% efficiency.     

Construction and Characterization of a Highly Efficient Francisella Shuttle Plasmid

Francisella tularensis is a facultative intracellular pathogen that infects a wide variety of mammals and causes tularemia in humans. It is recognized as a potential agent of bioterrorism due to its low infectious dose and multiple routes of transmission. To date, genetic manipulation in Francisella spp. has been limited due to the inefficiency of DNA transformation, the relative lack of useful selective markers, and the lack of stably replicating plasmids. Therefore, the goal of this study was to develop an enhanced shuttle plasmid that could be utilized for a variety of genetic procedures in both Francisella and Escherichia coli. A hybrid plasmid, pFNLTP1, was isolated that was transformed by electroporation at frequencies of >1 × 10⁷ CFU μg of DNA⁻¹ in F. tularensis LVS, Francisella novicida U112, and E. coli DH5α. Furthermore, this plasmid was stably maintained in F. tularensis LVS after passage in the absence of antibiotic selection in vitro and after 3 days of growth in J774A.1 macrophages. Importantly, F. tularensis LVS derivatives carrying pFNLTP1 were unaltered in their growth characteristics in laboratory medium and macrophages compared to wild-type LVS. We also constructed derivatives of pFNLTP1 containing expanded multiple cloning sites or temperature-sensitive mutations that failed to allow plasmid replication in F. tularensis LVS at the nonpermissive temperature. In addition, the utility of pFNLTP1 as a vehicle for gene expression, as well as complementation, was demonstrated. In summary, we describe construction of a Francisella shuttle plasmid that is transformed at high efficiency, is stably maintained, and does not alter the growth of Francisella in macrophages. This new tool should significantly enhance genetic manipulation and characterization of F. tularensis and other Francisella biotypes.     

Gene Transfer by Transduction in the Marine Environment

To determine the potential for bacteriophage-mediated gene transfer in the marine environment, we established transduction systems by using marine phage host isolates. Plasmid pQSR50, which contains transposon Tn5 and encodes kanamycin and streptomycin resistance, was used in plasmid transduction assays. Both marine bacterial isolates and concentrated natural bacterial communities were used as recipients in transduction studies. Transductants were detected by a gene probe complementary to the neomycin phosphotransferase (nptII) gene in Tn5. The transduction frequencies ranged from 1.33 × 10⁻⁷ to 5.13 × 10⁻⁹ transductants/PFU in studies performed with the bacterial isolates. With the mixed bacterial communities, putative transductants were detected in two of the six experiments performed. These putative transductants were confirmed and separated from indigenous antibiotic-resistant bacteria by colony hybridization probed with the nptII probe and by PCR amplification performed with two sets of primers specific for pQSR50. The frequencies of plasmid transduction in the mixed bacterial communities ranged from 1.58 × 10⁻⁸ to 3.7 × 10⁻⁸ transductants/PFU. Estimates of the transduction rate obtained by using a numerical model suggested that up to 1.3 × 10¹⁴ transduction events per year could occur in the Tampa Bay Estuary. The results of this study suggest that transduction could be an important mechanism for horizontal gene transfer in the marine environment.     

Heterologous Expression and Efficient Secretion of Chitosanase from Microbacterium sp. in Escherichia coli

A recombinant expression vector, pCT7-CHISP6H, was constructed for the secretory expression of mature peptide of chitosanase (mMschito) from Microbacterium sp. OU01. The vector contains several elements, including T7 promoter, signal peptide sequence of mschito, 6 × His-tag sequence and PmaCI restriction enzyme cloning site. In pCT7-CHISP6H, mMschito was fused into signal peptide sequence of mschito gene to construct recombinant plasmid pCT7-CHISP6H-mMschito. The recombinant plasmid was transformed into Escherichia coli BL21(DE3) and then expressed. The recombinant protein was secreted into the Luria–Bertani broth and the chitosanase activity in supernatant of the culture could reach up to 67.56 U/mL. The rmMschito in the broth supernatant was purified using HisTrap™ FF Crude column and the purified rmMschito was shown to be apparent homogeneity by 12 % SDS–PAGE analysis. Detected by 4700 MALDI-TOF–TOF-MS, the molecular weight of the purified rmMschito was 26,758.1875 and it was consistent with the predicted molecular weight. Chitosan (degree of deacetylation of 99 %) was mostly hydrolyzed into chitopentaose, chitotriose, and chitobiose by the purified rmMschito.     

IncP Plasmids Are Unusually Effective in Mediating Conjugation of Escherichia coli and Saccharomyces cerevisiae: Involvement of the Tra2 Mating System

Mobilizable shuttle plasmids containing the origin-of-transfer (oriT) region of plasmids F (IncFI), ColIb-P9 (IncI1), and RP4/RP1 (IncPα) were constructed to test the ability of the cognate conjugation system to mediate gene transfer from Escherichia coli to Saccharomyces cerevisiae. Only the Pα system caused detectable mobilization to yeast, giving peak values of 5 × 10⁻⁵ transconjugants per recipient cell in 30 min. Transfer of the shuttle plasmid required carriage of oriT in cis and the provision in trans of the Pα Tra1 core and Tra2 core regions. Genes outside the Tra1 core did not increase the mobilization efficiency. All 10 Tra2 core genes (trbB, -C, -D, -E, -F, -G, -H, -I, -J, and -L) required for plasmid transfer to E. coli K-12 were needed for transfer to yeast. To assess whether the mating-pair formation (Mpf) system or DNA-processing apparatus of the Pα conjugation system is critical in transkingdom transfer, an assay using an IncQ-based shuttle plasmid specifying its own DNA-processing system was devised. RP1 but not ColIb mobilized the construct to yeast, indicating that the Mpf complex determined by the Tra2 core genes plus traF is primarily responsible for the remarkable fertility of the Pα system in mediating gene transfer from bacteria to eukaryotes.     

Conjugative Transfer between Escherichia coli and Leptospira spp. as a New Genetic Tool

Our understanding of leptospiral pathogenesis, which remains poorly understood, depends on reliable genetic tools for functional analysis of genes in pathogenic strains. In this study, we report the first demonstration of conjugation between Escherichia coli and Leptospira spp. by using RP4 derivative conjugative plasmids. The DNA transfer described here was due to authentic conjugation, as shown by the requirement for cell-to-cell contact and the resistance of DNA transfers to the addition of DNase I. Transposition via conjugation of a plasmid delivering Himar1 yielded frequencies ranging from 1 × 10⁻⁶ to 8.5 × 10⁻⁸ transconjugants/recipient cell in the saprophyte L. biflexa and the pathogen L. interrogans, respectively. Analysis of mutants indicated that transposition occurs randomly, and at single sites in the genome of these strains, allowing the utilization of this system to generate libraries of transposon mutants.     

Generating In Vivo Cloning Vectors for Parallel Cloning of Large Gene Clusters by Homologous Recombination

A robust method for the in vivo cloning of large gene clusters was developed based on homologous recombination (HR), requiring only the transformation of PCR products into Escherichia coli cells harboring a receiver plasmid. Positive clones were selected by an acquired antibiotic resistance, which was activated by the recruitment of a short ribosome-binding site plus start codon sequence from the PCR products to the upstream position of a silent antibiotic resistance gene in receiver plasmids. This selection was highly stringent and thus the cloning efficiency of the GFPuv gene (size: 0.7 kb) was comparable to that of the conventional restriction-ligation method, reaching up to 4.3 × 10⁴ positive clones per μg of DNA. When we attempted parallel cloning of GFPuv fusion genes (size: 2.0 kb) and carotenoid biosynthesis pathway clusters (sizes: 4 kb, 6 kb, and 10 kb), the cloning efficiency was similarly high regardless of the DNA size, demonstrating that this would be useful for the cloning of large DNA sequences carrying multiple open reading frames. However, restriction analyses of the obtained plasmids showed that the selected cells may contain significant amounts of receiver plasmids without the inserts. To minimize the amount of empty plasmid in the positive selections, the sacB gene encoding a levansucrase was introduced as a counter selection marker in receiver plasmid as it converts sucrose to a toxic levan in the E. coli cells. Consequently, this method yielded completely homogeneous plasmids containing the inserts via the direct transformation of PCR products into E. coli cells.     

A Simple and Rapid Method for Genetic Transformation of Lactic Streptococci by Electroporation

An electroporation procedure for the plasmid-mediated genetic transformation of intact cells of Streptococcus cremoris and Streptococcus lactis was performed. Ten different strains were transformed. The method was simple and rapid and yielded transformant colonies in 14 to 24 h. The method was optimized for S. lactis LM0230, and transformation frequencies of between 1 × 10⁴ and 5 × 10⁵ transformants per μg of purified plasmid (pMU1328) were achieved routinely. The optimized procedure involved lysozyme treatment of cells. Transformation of LM0230 occurred at comparable frequencies with pLS1 (4.4 kilobase pair [kbp]), pMU1328 (7.4 kbp), and pAMβ1 (26.5 kbp). Plasmid DNA isolated from transformants had not undergone detectable deletions or rearrangements. Transformation was possible with plasmid DNA which was religated after restriction endonuclease digestion. Phage DNA-dependent transfection of S. lactis LM0230 and S. lactis C6 was also achieved.     

Transfer and Occurrence of Large Mercury Resistance Plasmids in River Epilithon

In situ mating experiments were done in the River Taff, South Wales, United Kingdom, by using a natural mercury resistance plasmid (pQM1) isolated from a mixture of epilithic bacteria in vitro. The river temperature from March to November was found to influence transfer frequencies strongly (6.8 × 10⁻⁹ to 1.5 × 10⁻² per recipient). A linear relationship existed between log₁₀ transfer frequency and river temperature (6 to 21°C), a 2.6°C change in temperature giving a 10-fold change in transfer frequency. In vitro experiments showed that pQM1 transferred most efficiently between fluorescent pseudomonads and that one epilithic isolate (Pseudomonas fluorescens) was an efficient donor in situ. Experiments with a P. putida recipient showed that intact epilithic bacterial communities could transfer mercury resistance plasmids in situ at frequencies of up to 3.75 × 10⁻⁶ per recipient. Nineteen of the large (>250-kilobase) plasmids isolated by transfer into P. putida were studied in detail and grouped into seven types by restriction digests. Mercury resistance and UV resistance were found to be common linked phenotypes in 19 of the 23 plasmids tested.     

Electron Microscopy of Viral RNA: Molecular Weight Determination of Bacterial and Animal Virus RNAs

A method for preparation of single-stranded RNA for electron microscopy determination of molecular weight is reported. The method uses treatment with formaldehyde at elevated temperatures to remove secondary structure and spreading in a protein monolayer from 50% formamide onto a 50% formamide hypophase. Molecular weights were determined for some bacterial and animal viruses, for which conflicting values had been reported earlier. Molecular weights determined by the method, using Escherichia coli large subunit rRNA for a standard (1.1 × 10⁶), are as follows: E. coli small subunit rRNA, 0.53 × 10⁶; coliphage f2-RNA, 1.3 × 10⁶; Qβ-RNA, 1.55 × 10⁶; and Newcastle disease virus RNA, 5.78 × 10⁶.