Introduction and maintenance of prokaryotic DNA inUstilago violacea

Summary A strain of the basidiomycete,Ustilago violacea, was transformed with a prokaryotic plasmid, pMP4-1, which confers resistance to neomycin.U. violacea transformants were selected at a frequency of 5 per g pMP4-1 DNA. Such transformants were at least 8-fold more resistant to neomycin than was the untransformed recipientU. violacea. Enzyme activity associated with the neomycin resistance gene was also found in the transformants. Southern DNA-DNA hybridization detected pMP4-1-derived sequences in both nuclear and mitochondrially-associated DNAs from transformants. The patterns of hybridization suggested integration of pMP4-1 sequences into the respective genomes. DNA from the nuclear fraction ofU. violacea transformants failed to produceE. coli transformants resistant to neomycin or to carbenicillin. In contrast, DNA from the mitochondrially-associated fraction inU. violacea transformants producedE. coli transformants resistant to neomycin. TheE. coli transformants contained a pMP4-1-derivative, pWP8, which was subsequently shown by Southern blot analysis to harborU. violacea mitochondrial DNA. Thus, a prokaryotic plasmid can be used to transform the eukaryoteU. violacea and acquire endogenous sequences from this organism.     

Plasmid mediated metal and antibiotic resistance in marinePseudomonas

Pseudomonas sp isolated from the Bay of Bengal (Madras coast) contained a single large plasmid (pMR1) of 146 kb. Plasmid curing was not successful with mitomycin C, sodium dodecyl sulfate, acridine orange, nalidixic acid or heat. Transfer of mercury resistance from marinePseudomonas toEscherichia coli occurred during mixed culture incubation in liquid broth at 10^–4 to 10^–5 ml^–1. However, transconjugants lacked the plasmid pMR1 and lost their ability to resist mercury. Transformation of pMR1 intoE. coli competent cells was successful; however, the efficiency of transformation (1.49×10² Hg^r transformants g^–1 pMR1 DNA) was low.E. coli transformants containing the plasmid pMR1 conferred inducible resistance to mercury, arsenic and cadmium compounds similar to the parental strain, but with increased expression. The mercury resistant transformants exhibited mercury volatilization activity. A correlation existed between metal and antibiotic resistance in the plasmid pMR1.     

Electroporation of Bradyrhizobium japonicum

Summary Electroporation offers a fast, efficient and reproducible way to introduce DNA into bacteria. We have successfully used this technique to transform two commercially important strains of Bradyrhizobium japonicum, the nitrogen-fixing soybean symbiont. Initially, electroporation conditions were optimized using plasmid DNA which had been prepared from the same B. japonicum strain into which the{imDNA was to b}e transformed. Efficiencies of 10⁵-10⁶ transformants/g DNA were obtained for strains USDA 110 and 61A152 with ready-to-use frozen cells. Successful electroporation of B. japonicum with plasmid DNA prepared from Escherichia coli varied with the E. coli strain from which the plasmid was purified. The highest transformation efficiencies (10⁴ transformants/g DNA) were obtained using DNA prepared from a dcm ^– dam ^– strain of E. coli. This suggests that routine isolation of DNA from an E. coli strain incapable of DNA modification should help in increasing transformation efficiencies for other strains of bacteria where DNA restriction appears to be a significant obstacle to successful transformation. We have also monitored the rate of spontaneous mutation in electroporated cells and saw no significant difference in the frequency of streptomycin resistance for electroporated cells compared to control cells.     

A stable and efficient transformation system for Butyrivibrio fibrisolvens OB156

A 9.5-kb shuttle vector capable of replication and selection in both Escherichia coli and Butyrivibrio fibrisolvens was constructed. Plasmid pUC118 provided replication functions and ampicillin resistance selection in E. coli. In B. fibrisolvens, replication was controlled by the native plasmid pRJF1 from strain OB156, and selectability was provided by a 3.5-kb fragment of plasmid pAM1 containing the erythromycin resistance gene. Optimum conditions for transformation were 15 kV/cm, 2 h recovery, and plating in an agar overlay on medium containing 10 g erythromycin/ml. Maximum efficiency was 1.1×10⁵ transformants per g plasmid DNA (average 3×10⁴), and restriction mechanisms reduced efficiency by a factor of 2×10². Nonselective growth for 200 generations gave no measurable loss of plasmid.     

Electroporation and stable maintenance of plasmid DNAs in a biocontrol strain of Pseudomonas syringae

Transformation efficiencies as high as 10⁷ transformants g^–1 DNA have been previously reported for pseudomonads using electroporation protocols established for E. coli with plasmid DNAs prepared from methylation proficient E. coli hosts. We report here a protocol for electroporation of plasmid DNAs into a biocontrol strain of Pseudomonas syringae which could not be electroporated by standard E. coli methods. Transformation efficiencies of 10⁷ or higher were obtained with DNA recovered from initial P. syringae transformation or with DNA prepared from methylation deficient E. coli. Both plasmids used in this study were stably maintained in the absence of selection for at least 50 generations.     

Cryptic plasmid pBf1 fromButyrivibrio fibrisolvens AR10: Its use as a replicon for recombinant plasmids

A cryptic 2.85 kb plasmid (pBf1) was isolated from the rumen bacteriumButyrivibrio fibrisolvens strain AR10, ampped with restriction endonucleases, and cleavage sites suitable for attachment toEscherichia coli plasmids were identified. AR10 was not able to be cured of pBf1 by growth at 42°C or in 0.25 g ampicillin/ml, but growth in 50 g acridine orange/ml for three culture passages produced cured colonies at a frequency of <1%. Chimeric plasmids were constructed by combining pBf1 with theE. coli plasmid pUC18, in addition to the clindamycin resistance gene fromBacteroides fragilis plasmid pDP1 (pCW2 and pCW3), or the CAT gene fromE. coli plasmid pKK232-8 (pCK1). For plasmid construction, pBf1 was cleaved at two alternative restriction sites to increase the likelihood that replication control sequences would remain functional in at least one of the plasmids. Electroporation of AR10 yielded transformant populations that clearly maintained the plasmids and that appeared to express the ampicillinase gene of pUC18, although transformants were not readily selectable with any of the three antibiotics. The suitability of pBf1 as a replicon on which to base the construction of shuttle vectors was demonstrated clearly, by persistence of plasmid pCW3 in the absence of selective pressure, and the addition of appropriate selection factors is expected to yield practical transformation vectors.     

High-frequency transformation ofLactobacillus casei with plasmid pHY300PLK by electroporation

To optimize the conditions for transformation ofLactobacillus casei ATCC 27092 cells with plasmid pHY300PLK, a shuttle vector forEscherichia coli andBacillus subtilis, by electroporation, we investigated the effects of the electrical parameters (voltage and resistance), the concentration of plasmid DNA, the cell age and density, the electroporation buffer, and other factors. Under optimal conditions of 2.0 kV, 100 ohm, and 25F, a transformation efficiency as high as 1.4×10⁷ transformants per g of plasmid DNA was obtained, with a survival rate of about 50%.L. casei YIT 9021, one of the PL-1 phage mutants of the ATCC 27092 strain, was also transformed with the same plasmid under optimal conditions. The transformants were confirmed to harbor the same intact plasmid molecules by agarose gel electrophoretic analysis.     

Cloning vector system forNocardia spp.

An electro-transformation system has been developed forNocardia asteroides andNocardia corallina by using aNocardia-Escherichia coli shuttle vector. The shuttle vector, named pCY104, was constructed by joining a 2.5-kb crypticN. asteroides plasmid pCY101 with theE. coli plasmid pIJ4625. The resistance genes for kanamycin, chloramphenicol, and thiostrepton on plasmid pCY104 were expressed inN. asteroides andN. corallina. The transformation method was optimized forN. asteroides, and transformation efficiency of 8×10⁴ transformants per g plasmid DNA was achieved routinely.     

Overproduction of E. coli xylose isomerase

Summary A promoterles DNA fragment containing theE. coli xylose isomerase gene and its ribosome binding site was ligated into a plasmid downstream from the strong P_L promoter. The plasmid was then used to transformE. coli strains containing a temperature-sensitive repressor (cI857). The transformants overproduced xylose isomerase when the repressor was thermally inactivated.     

Chromate resistance and reduction in Pseudomonas fluorescens strain LB300

Pseudomonas fluorescens LB300 is a chromateresistant strain isolated from chromium-contaminated river sediment. Chromate resistance is conferred by the plasmid pLHB1. Strain LB300 grew in minimal salts medium with as much as 1000 g of K₂CrO₄ ml^–1, and actively reduced chromate to Cr(III) while growing aerobically on a variety of substrates. Chromate was also reduced during anaerobic growth on acetate, the chromate serving as terminal electron acceptor. P. fluorescens LB303, a plasmidless, chromatesensitive variant of P. fluorescens LB300, did not grow in minimal salts medium with more than 10 g of K₂CrO₄ ml^–1. However, resting cells of strain LB303 grown without chromate reduced chromate as well as strain LB300 cells grown under the same conditions. Furthermore, resting cells of chromate-sensitive Pseudomonas putida strain AC10, also catalyzed chromate reduction. Evidently chromate resistance and chromate reduction in these organisms are unrelated. Comparison of the rates of chromate reduction by chromate grown cells and cells grown without chromate indicated that the chromate reductase activity is constitutive. Studies with cell-free extracts show that the reductase is membrane-associated and can mediate the transfer of electrons from NADH to chromate.     

The ChrA homologue from a sulfur-regulated gene cluster in cyanobacterial plasmid pANL confers chromate resistance

The cyanobacterium Synechococcus elongatus strain PCC 7942 possesses pANL, a plasmid rich in genes related to sulfur metabolism. One of these genes, srpC, encodes the SrpC protein, a homologue of the CHR chromate ion transporter superfamily. The srpC gene was cloned and expressed in Escherichia coli and its role in relation to sulfate and chromate was analyzed. srpC was unable to complement the growth of an E. coli cysA sulfate uptake mutant when sulfate was utilized as a sole sulfur source, suggesting that SrpC is not a sulfate transporter. Expression of srpC in E. coli conferred chromate resistance and caused diminished chromate uptake. These results suggest that the S. elongatus SrpC protein functions as a transporter that extrudes chromate ions from the cell’s cytoplasm, and further demonstrate the close relationship between sulfate and chromate metabolism in this organism.     

Improvement of plasmid encoded lactococcal bacteriophage resistance by mutator strain Epicurian coli XL1-Red

A random mutation strategy using mutator strain, Epicurian coli XL1-Red, was applied to a plasmid, pND018, constructed by inserting a Lactococcus lacis bacteriophage resistance gene (abiI) into a L. lactis/E. coli shuttle vector (pDL278), to introduce random mutations throughout the plasmid. Following transformation of the mutated plasmid library to a plasmid free and phage sensitive strain of L. lactis (LM0230), mutated plasmids were screened by cross-streaking and efficiency of plaquing (EOP) assays. Two strains with enhanced resistance were obtained, as well as several phage sensitive strains. Repeated transformation of the mutated plasmids to LM0230 confirmed that the observed phenotypes were caused by mutations located on the plasmids. The EOP values and plaque morphology of two enhanced phage resistance mutants were characterized at 30°C and 37°C. These results indicate that this simple procedure can be applied to generate modified plasmids with improved phage resistance, which may be of commercial value.     

Mercury Volatilization by R Factor Systems in <Emphasis Type="Italic">Escherichia coli</Emphasis> Isolated from Aquatic Environments of India

Ten Escherichia coli strains isolated from five different aquatic environments representing three distinct geographical regions of India showed significantly high levels of tolerance to the inorganic form of mercury, i.e., mercuric chloride (HgCl₂). MRD14 isolated from the Dal Lake (Kashmir) could tolerate the highest concentration of HgCl₂, i.e., 55 g/mL, and MRF1 from the flood water of the Yamuna River (Delhi) tolerated the lowest concentration, i.e., 25 g/mL. All ten strains revealed the presence of a plasmid of approximately 24 kb, and transformation of the isolated plasmids into the mercury-sensitive competent cells of E. coli DH5 rendered the transformants resistant to the same concentration of mercury as the wild-type strains. Mating experiments were performed to assess the self-transmissible nature of these promiscuous plasmids. The transfer of mercury resistance from these wild-type strains to the mercury-sensitive, naladixic acid-resistant E. coli K12 (F^–lac⁺) strain used as a recipient was observed in six of the nine strains tested. Transconjugants revealed the presence of a plasmid of approximately 24 kb. An evaluation of the mechanism of mercury resistance in the three most efficient strains (MRG12, MRD11, and MRD14) encountered in our study was determined by cold vapor atomic absorption spectroscopy (CV-AAS), and it was noted that resistance to HgCl₂ was conferred by conversion of the toxic ionic form of mercury (Hg⁺⁺) to the nontoxic elemental form (Hg⁰) in all three strains. MRD14 volatilized mercury most efficiently.     

Construction of a new host-vector system inArthrobacter sp. and cloning of the lipase gene

Arthrobacter sp. strain MIS38 was transformed with a shuttle vector containing the kanamycin resistant genekan (derived from Tn5) by an electroporation method. This shuttle vector is fromBrevibacterium lactofermentum andEscherichia coli, pULRS8: - The following optimal condition of electroporation was determined. A square wave pulse of 1 kV/cm electric field strength for 0.5 ms duration yielded 3 × 10⁵ transformants/,g plasmid DNA. The number of transformants increased with the amount of DNA over the range 0.01-5 g. This host-vector system was then used successfully to clone and express a lipase gene fromArthrobacter sp. strain MIS38 into bothArthrobacter sp. MIS38 and E. coli JM109.     

Quantitative detection of asbestos fiber in gravelly sand using elastic body-exposure method

Chrysotile or crocidolite colloidal solution containing donor plasmid DNA and Escherichia coli cells was subjected to elastic body friction. These acicular clay minerals mediated E. coli antibiotic resistance plasmid transformation. Other clay minerals had no effect on E. coli transformation. The number of E. coli transformants was counted after elastic body exposure with various crocidolite concentrations. There was a correlation between the number of E. coli transformants and crocidolite concentration (between 40 and 1,000 ng/ml). A mixture consisting of sea sand and crocidolite was utilized as a model for quantitative detection of asbestos in gravelly sand. With sea sand containing 0.15–15 mg of crocidolite, a correlation between crocidolite concentration and the number of colonies derived from E. coli transformants was observed. This indicates that measurement of asbestos is possible even when the asbestos sample includes gravelly sand. Fluorescence microscopic observation of crocidolite colloidal solution indicated that crocidolite was present as spherical aggregates having diameters of 6–9 μm. Thus, the number of transformants correlated with that of 6–9 μm crocidolite aggregates.     

Plasmid vectors used in various strains of Erwinia species

Summary Two multiple-copy plasmid vectors, pBEH3-5 and pBEH8-2 were constructed from a Erwinia plasmid, pEC3 or pEC8, and the Escherichia coli plasmid pBR328. Part of sequence homology between pEC3 and pEC8 was found by Southern hybridization. The two vectors were efficiently transferred into members of the species E. amylovora, E. carotovora, E. carotovora subsp. carotovora, and E. herbicola using a binary plasmid system with RP4. The transformation system examined in strains of these Erwinia species yields about 2 to 4x10² transformants per g of pBEH3-5 DNA. These host-vector systems make potentially useful tools for the study of genes involved in the plant pathogenesis of Erwinia species.     

Cloning and expression of Citrobacter freundii β-isopropylmalate dehydrogenase gene in both Escherichia coli and Bacillus subtilis

Summary -Isopropylmalate (IPM) dehydrogenase gene of Citrobacter freundii was cloned in both Escherichia coli and Bacillus subtilis. Plasmid pCBL 1 containing C. freundii -IPM dehydrogenase gene was isolated using E. coli (leuB) as a host, pBR 322 as a vector and Hind III as an enzyme. The molecular weight (mol.wt.) of pCBL 1 was 7.7 megadalton (Md) and the plasmid was restricted at two sites by Hind III or Sal I, at three sites by BamH I and at four sites by Pst I. The second hybrid plasmid pCBL 2 containing -IPM dehydrogenase gene was reconstructed from 2.1 Md Pst I fragment of pCBL 1 and pBR 322. -IPM dehydrogenase activities of E. coli transformants with pCBL 1 or pCBL 2 were 2–7-fold higher than those of the present strains. The -IPM dehydrogenase gene was transferred from pBR 322 to pLS 353, a shuttle vector between E. coli and B. subtilis. The third plasmid, pCBL 3 (mol.wt. 5.6Md), was cloned in B. subtilis (leuC) and expressed the enzyme activity which complemented the Leucharacter. The enzyme activities of B. subtilis transformants with pCBL 3 were about 5-fold higher than those of present strains. Thus, the C. freundii gene was effectively expressed in both E. coli and B. subtilis.     

A bifunctional plasmid carrying the recA gene of E. coli

Summary To investigate the effect of an active, plasmid-carried recA gene on the stability and/or the expression of plasmid genes in different genetic backgrounds, we have constructed a bifunctional plasmid (able to replicate in Escherichia coli and in Bacillus subtilis). Chimeric plasmids were obtained by inserting pC194 (Ehrlich 1977) into pDR1453 (Sancar and Rupp 1979). pDR1453 is a 12.9 Kbp plasmid constructed by inserting an E. coli chromosome fragment carrying the recA gene into pBR322. The expected bifunctional recombinant (pMR22/1) (15.7 Kbp) was easily obtained but surprisingly the Cm resistance was expressed only at a very low level in E. coli (as compared, for example, to pHV14, pHV15). We attribute this effect to the presence of multiple recA genes in the cell. On the contrary, Cm^r E. coli transformants bear a recombinant plasmid (pMR22/n) containing tandemly repeated copies of pC194 in equilibrium with excised free pC194. Such amplification has never been observed in a Rec^- background and is therefore mediated by the recA genes. Growth of these clones in the absence of Cm causes the loss of the extra copies, yielding a plasmid with a single copy of pC194, indistingishable from pMR22/1. Interestingly, we have observed that deletions occur at high frequency in pC194, which drastically increase Cm^r in E. coli containing plasmids with a single copy of pC194. Two types of such deletions were detected: (a) large 1050 bp deletions covering about onethird of pC194 and (b) small 120–150 bp deletions (near the MspI site) in the region containing the replicative functions of pC194 (Horinouchi and Weisblum 1982). Both types of deletion render the recombinant plasmid unable to replicate in B. subtilis. pM22/1 replicates, although with a low copy-number, and is stable in B. subtilis wild type; the recA gene of E. coli does not complement any of the rec ^- mutations of B. subtilis. A strong instability, mainly of the E. coli and pBR322 sequences, was observed in many dna and rec mutants of B. subtilis yielding smaller plasmid with a much higher copy-number.     

Development of an electro-transformation system for Escherichia coli DH10B

Summary Escherichia coli can be transformed to high efficiencies by subjecting a mixture of cells and DNA to a brief but intense electrical field. Factors that affect the transformation efficiency of E.coli strain DH10B were analysed. Optimal conditions gave an efficiency of 10⁸ to 10⁹ transformants/g DNA with E.coli strains K803 and DH10B, and plasmids pB1221.23 and pBSK+. The use of ligated DNA resulted in 10⁶ transformants/g DNA. Detailed protocols for these systems are given.